WO2025035137A2 - Synthetic helper genes for enhancing viral production - Google Patents

Abstract

The present disclosure provides methods and compositions for production of recombinant viral vectors, such as adeno-associated virus (AAV) vectors or lentiviral vectors, in host cells using Synthetic Helper Gene Products that increase viral titer and transduction efficiency of viral vector compositions. The present disclosure also provides methods for selecting genetically encoded, endogenously expressed Synthetic Helper Gene Products that enhance viral vector manufacturability.

Description

SYNTHETIC HELPER GENES FOR ENHANCING VIRAL PRODUCTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Patent Application No. 63/518,812 filed August 10, 2023, entitled “SYNTHETIC HELPER GENES FOR ENHANCING VIRAL PRODUCTION,” which is herein incorporated by reference in its entirety for all purposes.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[002] The contents of the electronic sequence listing (SYNV02PCT_SEQLIST.xml; Size: 322,665 bytes, and Date of Creation: August 8, 2024) is herein incorporated by reference in its entirety.

TECHNICAL FIELD

[003] The present disclosure generally relates to biotechnology and, in particular, to methods and compositions for production of recombinant viral vectors, such as AAV vectors, in host cells. Recombinant viral vectors can be used as gene delivery vehicles for treatment of human diseases.

INTRODUCTION

[004] Production of highly infectious viral vector compositions has significant challenges. For example, recombinant adeno-associated virus (AAV) is a leading gene delivery platform for treatment of human diseases with many advantages, including good safety profile, long persistence of AAV-delivered genetic payloads in target cells, as well as strong and diverse tropism (ability to target specific tissues). However, AAV production at an industrial scale has been accomplished only to a limited degree. There are several major problems with the manufacturing and use of AAV vectors as gene therapy. When using helper virus strategies, the resulting AAVs are highly contaminated with pathogenic viruses, which can be challenging to remove. For this reason, helper virus free approaches are typically preferred. However, different challenges arise. First, up to 90% of viral capsids produced during manufacturing can be empty. Although it is possible to purify full capsids away from empty capsids, this procedure is costly, difficult to scale, and reduces yield. Second, among the properly packaged recombinant viral capsids, only a small fraction of them is able to deliver their payload into target cells (often 0.1% or less). To compensate for this during treatment, the dose needs to be increased significantly; however, this increases the cost of treatment and reduces the safety profile.

[005] A cause of these problems is that the intracellular environment of packaging cells is not optimized for producing viral vectors. For example, it is known that during the AAV natural replication cycle, the associated adenovirus significantly perturbs and optimizes the cellular environment as part of its own lifecycle, which also provides a cellular milieu that is highly optimized for AAV replication. Since the use of “helper” adenoviruses to assist AAV production poses a significant safety risk, current AAV manufacturing platforms avoid the use of helper viruses instead relying on cloned helper genes. Expression of cloned helper genes poorly mimics the adenovirus induced changes to the intracellular environment and may have undesirable effects on the host cell used to package AAV vectors. The multitude of mechanisms that determine AAV vector quality are not well understood. Post-translational modifications, such as glycosylation, acetylation, phosphorylation of AAV capsid, as well as variable DNA methylation of the AAV genome, are all thought to play important roles.

[006] There is a need for improved viral production methods that will satisfy the current demand of viral vector material for clinical trials and market supply. The current invention addresses this need by providing methods of production of highly infectious viral vector compositions.

[007] These and other embodiments of the invention will be apparent upon reference to the following detailed description. To this end, various references are set forth herein which describe in more detail certain background information, procedures, compounds and/or compositions, and are each hereby incorporated by reference in their entireties.

SUMMARY

[008] The summary is not intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the detailed description including those embodiments disclosed in the accompanying drawings and in the appended claims.

[009] The present teachings include methods that allow the discovery of endogenously expressed Synthetic Helper Gene Products that can positively modulate/enhance viral packaging and manufacturability. The Synthetic Helper Gene platform is used to optimize cell behavior for viral production. Synthetic Helper Gene Products enhance viral vector production when expressed in producer cells. Unlike helper genes derived from other natural sources, Synthetic Helper Genes are artificial, synthetic constructs (i.e. not merely naturally occurring or unmodified genes). The proposed methods also improve the infectiousness of the produced viral vector composition.

[0010] Synthetic Helper Genes offer enhanced engineerability, providing more efficient means of enhancing compatibility between host cells and viral systems. In a certain regard, they can be considered a highly engineerable adapter layer that allows packaging cell and viral biology to more effectively interface in order to optimize viral production. For example, a Synthetic Helper Gene could encode a novel protein that interacts with viral components to improve capsid assembly or stability.

[0011] Synthetic Helper Genes encode Synthetic Helper Gene Products. Synthetic Helper Gene Products are short protein products and can take two forms including antibodylike proteins (e.g. nanobodies) and engineered transcription factors. These Synthetic Helper Gene Products have a capacity to manipulate biological systems through interactions with various biological molecules within in the cell (e.g. proteins for antibody-like proteins and DNA for transcription factors). The key features are that they are artificial or synthetic polypeptides that are expressed in viral packaging cells during viral production so that the synthetic/ engineered binding profile results in a perturbation to the cell, the result of which is to enhance viral production (e.g. with respect to yield or viral infectiousness). Of particular import is that the activity of SHGPs is limited to the viral production process and is not essentially present in the resulting viral product (e.g. as in antibody-functionalized or mutated capsids) or in the use of the viral product (e.g. as in small molecule, peptide, or protein-based transduction enhancers).

[0012] One way they could interfere with biological functions is through direct inhibition. For instance, a nanobody SHGP may bind to a target protein, thereby blocking its active site. This obstructive presence can prevent the protein from performing its desired functions effectively.

[0013] Another route of manipulation is through allosteric regulation. Instead of directly blocking the active site, a nanobody may bind to a non-active site on the protein. This binding can induce a conformational change in the protein structure, thereby reducing its activity or even rendering it inactive.

[0014] Synthetic Helper Gene Products can also indirectly inhibit biological functions. For example, antibody-like proteins may drive aggregation. This clumping together can interfere with their normal functions and sequester them.

[0015] In addition, these products may alter the production or prevention of protein drives of biological functions. Certain antibody-like proteins may bind to proteins in a way that hampers their proper folding or stability. This can potentially result in a loss of function.

[0016] Moreover, they can modify the turnover dynamics of their target proteins. For instance, an antibody-like may be translationally fused to a second effector domain, resulting in the target protein's destruction, as seen in the case of ubiquitination. Conversely, a binding domain may be connected to a second effector domain that halts the destruction of the target protein, as in the case of deubiquitinase.

[0017] Transcription factor SHGPs may alter gene expression by acting as synthetic and orthogonal transcriptional regulators. By binding to one or more sites in the cell, DNA expression, and thus cell state, is expected to be perturbed, with the resulting perturbation manifesting in a change in viral production performance.

[0018] Despite the extensive versatility of Synthetic Helper Gene Products, we have identified two classes that are particularly effective for manipulating cell behavior to optimize viral production: engineered transcription factors and antibody-like proteins. The latter category includes antibody mimetics (e.g. affibodies, DARPINs) and single domain antibodies (e.g. nanobodies, scFv).

[0019] In one embodiment, the present invention pertains to the use of Synthetic Helper Genes for enhancing the production of recombinant Adeno- Associated Virus (rAAV). The Synthetic Helper Genes encode proteins or peptides that modulate or interfere with various known cellular and viral responses that impact rAAV production, offering significant advancements in the field. These embodiments, however, are non-limiting and merely exemplary of the broad application of the invention.

[0020] In one non-limiting example, Synthetic Helper Gene Products are synthetic to modulate cellular innate antiviral pathways. They may inhibit pathways such as the RIG- I/MDA5/0AS1, interferons, interferon-stimulated genes (ISGs), inflammasomes, cytokines, and IRFs, which have been reported to inhibit AAV production. Moreover, they may interfere with the cGAS/STING pathway and pattern recognition receptors, which restrict AAV replication and production through their antiviral responses.

[0021] In another non-limiting example, Synthetic Helper Gene Products are designed to counteract the adverse effects of helper genes and AAV genes on cell health and productivity. The proteins encoded by the Synthetic Helper Genes can counter the induction of cell cycle changes and DNA damage responses triggered by adenovirus E1A/E1B and E4 genes, commonly used in AAV production. They may also counter the effects of the AAV Rep78 protein, known to induce cell cycle arrest and apoptosis.

[0022] Synthetic Helper Gene Products can also be utilized to alleviate cellular stress pathways, such as MAPK signaling and DNA damage responses, which are activated during AAV production and negatively affect cell health and productivity.

[0023] The Synthetic Helper Genes can additionally encode products that modify AAV capsid properties to enhance vector function. These modifications include, but are not limited to, acetylation, methylation, phosphorylation, O-GlcNAcylation, glycosylation, deamidation, ubiquitination, sumoylation, proteolysis, and pH processing.

[0024] In yet another non-limiting example, Synthetic Helper Gene Products can be employed to alter the packaged genome and host cell DNA, thus enhancing the effectiveness of AAV vectors. They may modulate CpG methylation, a known factor affecting gene expression in AAV vectors and thereby their potency.

[0025] Furthermore, Synthetic Helper Gene Products can regulate aspects of the cell substrate to impact the yield, safety, and potency of AAV vectors, such as the formation of empty capsids and the presence of host cell protein impurities.

[0026] In summary, the present invention pertains to the utilization of Synthetic Helper Genes encoding proteins or peptides capable of modulating a wide array of cellular and viral responses to enhance rAAV production. The embodiments described herein represent a non- exhaustive list of the many possible applications of these Synthetic Helper Genes, which may extend to additional pathways and processes not currently detailed in the existing literature. As such, the full scope of the invention is not limited to these examples.

[0027] The present invention also extends to the use of Synthetic Helper Genes for enhancing the production of recombinant lentiviral vectors. The Synthetic Helper Genes are capable of encoding proteins or peptides that interact with various known and unknown cellular and viral responses, to improve lentiviral production efficiency. The embodiments described herein serve as non-limiting examples of the numerous possible ways in which Synthetic Helper Genes can be applied to enhance lentiviral production.

[0028] Lentiviral vectors, like AAV vectors, are susceptible to innate cellular antiviral responses that can impact their production. While the exact mechanisms and pathways involved may differ between these vector types, the general concept of using Synthetic Helper Genes to modulate these responses remains applicable. For instance, Synthetic Helper Genes might be designed to inhibit or modulate responses triggered by pattern recognition receptors or other cellular antiviral pathways that can inhibit lentiviral production.

[0029] Furthermore, lentiviral vectors, much like AAV vectors, can induce cellular stress responses that affect cell health and productivity. Synthetic Helper Gene Products can potentially alleviate these stress responses, enhancing the cellular environment for lentiviral production. Additionally, Synthetic Helper Genes might encode proteins that are able to interact with, and modulate, lentiviral genes or proteins that have adverse effects on cell health and productivity. [0030] Moreover, Synthetic Helper Genes can also be utilized to modify the properties of lentiviral capsids or other vector components to enhance vector function. For example, Synthetic Helper Genes might encode proteins or peptides that modify the glycosylation patterns or other post-translational modifications of lentiviral envelope proteins, which could impact vector infectivity or host immune responses.

[0031] As with AAV vectors, Synthetic Helper Genes could also be used to influence the packaged genome and host cell DNA to enhance the potency of lentiviral vectors. Furthermore, Synthetic Helper Genes could help regulate the formation of defective interfering particles or other aspects of the lentiviral production process to enhance the yield and quality of lentiviral vectors.

[0032] In summary, the Synthetic Helper Genes offer a versatile tool for improving lentiviral vector production. While the specific pathways and processes described for AAV may not apply directly to lentiviral vectors, the underlying concept of using Synthetic Helper Genes to manipulate cellular and viral responses is broadly applicable. As such, the examples provided here serve as non-limiting embodiments of the invention's application in the context of lentiviral vector production. The full scope of the invention extends to numerous other potential applications, some of which may not currently be detailed in the existing literature. [0033] The present invention's application is not limited to adeno-associated virus (AAV) or lentiviral vectors but extends to enhancing the production of any viral vectors. The Synthetic Helper Genes can encode proteins or peptides designed to modulate cellular and viral pathways to optimize the production of a variety of viral vectors. The described embodiments serve as non-limiting examples, as Synthetic Helper Genes can potentially interact with a diverse range of known and possibly unknown pathways in different types of viruses.

[0034] In general, all viruses are susceptible to the innate antiviral defense mechanisms of the host cells, which may include various cellular antiviral responses, pattern recognition receptors, interferons, and more. Though the specifics of these responses may vary depending on the type of virus, Synthetic Helper Genes can potentially be designed to modulate these responses across different viral systems, enhancing viral vector production.

[0035] Similarly, Synthetic Helper Genes can potentially be utilized to modulate cellular stress responses induced by viral replication across various viral systems. Even though these stress responses may vary depending on the virus type and cell type involved, Synthetic Helper Genes could still potentially alleviate these stress responses and enhance the environment for viral production. [0036] Additionally, Synthetic Helper Genes can be utilized to modulate the properties of the viral proteins in a variety of viral systems. This could include the modification of post- translational modifications on viral capsid or envelope proteins, which could alter the viral vector's properties and affect their functionality.

[0037] Synthetic Helper Genes might also influence the packaged viral genome and host cell DNA across various viral systems to enhance the potency of the resulting viral vectors. Furthermore, they could also assist in controlling the formation of defective interfering particles or other phenomena that could affect the yield and quality of viral vectors in different viral systems.

[0038] In conclusion, the concept of Synthetic Helper Genes offers a broad-spectrum tool for improving the production of any viral vectors, beyond just AAV or lentiviral systems. The described embodiments are non-limiting, and the full scope of the invention extends to numerous other potential applications and interactions with various cellular and viral responses, some of which may not currently be detailed in the existing literature.

[0039] The present teachings include a method of obtaining a Synthetic Helper Gene (SHG) encoding a Synthetic Helper Gene Product (SHGP) which can increase viral titer and/or transduction efficiency of a viral vector composition, the method comprising:

(a) culturing a first plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary for the production of the virus in the host cells; and

(iv) a Synthetic Helper Gene Product comprising an polypeptide encoded by a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to one or more one or more viral-specific packaging sequences necessary for encapsulation of the first nucleotide sequence within the viral capsids or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to the one or more one or more viral-specific packaging sequences necessary for encapsulation of the second nucleotide sequence within the viral capsids, thereby obtaining a first plurality of viral vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of viral vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for replication of the virus; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences producing the Synthetic Helper Gene Product, thereby obtaining a plurality of viral vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of viral vectors or the plurality of viral vectors of the present cycle to infect a final plurality of host cells; and (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of the viral vector composition by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from (i) the final plurality of host cells and/or (ii) a final plurality of viral vectors produced in the final plurality of host cells.

[0040] The present teachings also include a plurality of host cells permissive for replication of a virus, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary for the production of the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; wherein the Synthetic Helper Gene Product increases a characteristic of viral vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of viral vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of viral vectors is selected from the group consisting of viral titer and transduction efficiency. [0041] The present teachings also include a method of producing a viral vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(a) culturing a plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; and (b) producing the viral vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the viral vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral titer and/or transduction efficiency of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

[0042] The present teachings also include a method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of adeno- associated virus (AAV) vector composition, the method comprising:

(a) culturing a first plurality of host cells permissive for AAV replication under conditions suitable for recombinant AAV production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one AAV replication protein produced from at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

(ii) at least one AAV capsid encoding protein produced from at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15);

(iii) at least one AAV helper protein produced from at least one AAV helper gene (SEQ ID NO: 16-23); and

(iv) a Synthetic Helper Gene Product produced from a Synthetic Helper Gene encoded by a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to at least two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to at least two functional AAV ITRs, thereby obtaining a first plurality of AAV vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of AAV vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for AAV replication; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant AAV production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the at least two functional AAV ITRs producing the Synthetic Helper Gene Product, thereby obtaining a plurality of AAV vectors of the present cycle comprising the first nucleotide sequence; (c) allowing the first plurality of AAV vectors or the plurality of AAV vectors of the present cycle to infect a final plurality of host cells; and (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of AAV vector composition by analyzing nucleotide sequences operably linked to the at least two functional AAV inverted terminal repeats (ITRs) from (i) the final plurality of host cells and/or (ii) a final plurality of AAV vectors produced in the final plurality of host cells.

[0043] The present teachings also include a method of producing an adeno-associated virus (AAV) vector composition, the method comprising:

(a) culturing a first plurality of host cells permissive for AAV replication under conditions suitable for recombinant AAV production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one AAV replication protein produced from at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

(ii) at least one AAV capsid encoding protein produced from at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15);

(iii) at least one AAV helper protein produced from at least one AAV helper gene (SEQ ID NO: 16-23); and

(iv) a Synthetic Helper Gene Product produced from a Synthetic Helper Gene encoded by a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to at least two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to at least two functional AAV inverted terminal repeats (ITRs), thereby obtaining a first plurality of AAV vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of AAV vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for AAV replication; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant AAV production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the at least two functional AAV ITRs producing the Synthetic Helper Gene Product, thereby obtaining a plurality of AAV vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of AAV vectors or the plurality of AAV vectors of the present cycle to infect a final plurality of host cells;

(d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of AAV vector composition by analyzing nucleotide sequences operably linked to the at least two functional AAV inverted terminal repeats (ITRs) from (i) the final plurality of host cells and/or (ii) a final plurality of AAV vectors produced in the final plurality of host cells; and (e) obtaining new AAV vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the AAV vector composition

(e.g., an adeno-associated virus (AAV) vector composition of increased viral titer and/or transduction efficiency).

[0044] The present teachings also include a plurality of host cells permissive for AAV replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one AAV replication protein produced from at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

(ii) at least one AAV capsid encoding protein produced from at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15); (iii) at least one AAV helper protein produced from at least one AAV helper gene (SEQ ID NO: 16-23); and

(iv) a nucleotide sequence operably linked to at least two functional AAV internal terminal repeats (ITRs) (SEQ ID NO: 8-9), wherein the nucleotide sequence encodes a payload (e.g. therapeutic gene); wherein the Synthetic Helper Gene Product increases a characteristic of AAV vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of AAV vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of AAV vectors is selected from the group consisting of physical titer, biological titer and transduction efficiency.

[0045] The present teachings also include a method of producing an adeno-associated virus (AAV) vector composition of increased viral titer and/or transduction efficiency, the method comprising: culturing a plurality of host cells permissive for AAV replication under conditions suitable for recombinant AAV production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one AAV replication protein produced from at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

(ii) at least one AAV capsid encoding protein produced from at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15);

(iii) at least one AAV helper protein produced from at least one AAV helper gene (SEQ ID NO: 16-23); and

(iv) a nucleotide sequence operably linked between two functional AAV internal terminal repeats (ITRs) (SEQ ID NO: 8-9), wherein the nucleotide sequence encodes a payload; and (b) producing the AAV vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the AAV vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than an viral titer and/or transduction efficiency of a reference AAV vector composition produced from a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product. [0046] The present teachings also include an AAV vector composition of increased viral titer and/or transduction efficiency produced by the disclosed methods.

[0047] In some embodiments, TU:VG ratio of the AAV vector composition of increased viral titer and/or transduction efficiency is from 1 : 100 to 1 :50, from 1 : 50 to 1 :20, from 1 :20 to 1 : 10, from 1 : 10 to 1 :5, from 1 :5 to 1 :2, or from 1 :2 to 1 : 1.

[0048] In preferred embodiments, each host cell is a mammalian cell or an insect cell. [0049] In some embodiments, the AAV vector composition of increased viral titer and/or transduction efficiency has a viral genome titer which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral genome titer of a reference AAV vector composition produced without Synthetic Helper Gene Products.

[0050] The present teachings also include a method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of lentivirus vector composition, the method comprising:

(a) culturing a first plurality of host cells permissive for lentivirus replication under conditions suitable for recombinant lentivirus production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one lentiviral gag gene (SEQ ID NO: 26);

(ii) at least one lentiviral pol gene (SEQ ID NO: 25);

(iii) at least one lentiviral rev gene (SEQ ID NO: 24);

(iv) at least one env gene (SEQ ID NO: 27); and

(v) a Synthetic Helper Gene Product produced from a Synthetic Helper Gene encoded by a first nucleotide sequence, wherein (vi) the first nucleotide sequence is operably linked to at a Psi sequence (SEQ ID NO: 34) or (vii) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to a Psi sequence (SEQ ID NO: 34), thereby obtaining a first plurality of lentivirus vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of lentivirus vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for lentivirus replication; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant lentivirus production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iv) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to a Psi sequence (SEQ ID NO: 34) producing the Synthetic Helper Gene Product, thereby obtaining a plurality of lentivirus vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of lentivirus vectors or the plurality of lentivirus vectors of the present cycle to infect a final plurality of host cells; and (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of lentivirus vector composition by analyzing nucleotide sequences operably linked to a Psi sequence (SEQ ID NO: 34) from (i) the final plurality of host cells and/or

(ii) a final plurality of lentivirus vectors produced in the final plurality of host cells.

[0051] The present teachings also include a plurality of host cells permissive for lentivirus replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one lentiviral gag gene (SEQ ID NO: 26);

(ii) at least one lentiviral pol gene (SEQ ID NO: 25);

(iii) at least one lentiviral rev gene (SEQ ID NO: 24);

(iv) at least one env gene (SEQ ID NO: 27); and

(v) a nucleotide sequence operably linked to a Psi sequence (SEQ ID NO: 34), wherein the nucleotide sequence encodes a payload; wherein the Synthetic Helper Gene Product increases a characteristic of lentivirus vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of lentivirus vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(v) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of lentivirus vectors is selected from the group consisting of viral titer and transduction efficiency.

[0052] The present teachings also include a method of producing a lentivirus vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(a) culturing a plurality of host cells permissive for lentiviral replication under conditions suitable for recombinant lentiviral production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one lentiviral gag gene (SEQ ID NO: 26);

(ii) at least one lentiviral pol gene (SEQ ID NO: 25);

(iii) at least one lentiviral rev gene (SEQ ID NO: 24);

(iv) at least one env gene (SEQ ID NO: 27); and (v) a nucleotide sequence operably linked to a Psi sequence (SEQ ID NO: 34), wherein the nucleotide sequence encodes a payload (e.g. therapeutic gene); and (b) producing the lentivirus vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the lentivirus vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral titer and/or transduction efficiency of a reference lentivirus vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(v) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

[0053] The present teachings also include a lentivirus vector composition of increased viral titer and/or transduction efficiency produced by the disclosed methods.

[0054] These and other features, aspects and advantages of the present teachings will become better understood with reference to the following description, examples and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Those with skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0056] Fig. 1. Exemplary discovery of Synthetic Helper Gene Products that enhanced viral production with genetic selection and screening.

[0057] Fig. 1 Part A. Packaging cells were transfected with a library of genes configured to produce Synthetic Helper Gene Products and flanked by two ITRs (SEQ ID NO: 8-9). The library may also include additional elements: fluorescent proteins (such as green fluorescent protein or GFP), reporter enzymes (luciferase) or a barcode. The packaging cells contained DNA encoding Rep (SEQ ID NO: 1-4, 10, 12, 14), Cap (SEQ ID NO: 5-7, 11, 13, 15), and helper genes (SEQ ID NO: 16-23)(e.g. from triple transfection, stable integration, etc. . .). The Synthetic Helper Gene Product plasmid library (pSHG) is depicted as circular plasmids 1, 2, . . . , and N.

[0058] Fig. 1 Part B. The Synthetic Helper Gene Product library and genes required for AAV production were expressed in host cells (e.g. through triple transfection, stable genome integration, helper virus, etc. . .). Synthetic Helper Gene Products that reduced AAV production reduced their own coding DNA from being packaged. Synthetic Helper Gene Products that enhanced AAV production enhanced the packaging of their own coding DNA into AAV virions. A cartoon of petri dish with packaging cells is depicted.

[0059] Fig. 1 Part C. The library of AAV virions were harvested from the AAV packaging cells. The AAV library harbors DNA encoding Synthetic Helper Gene Products or DNA barcodes that was used to identify the Synthetic Helper Gene Product present inside of the cell that the AAV was produced in. The library population is enriched for Synthetic Helper Gene Product variants (or corresponding DNA barcodes) that enhance AAV titer (black AAV capsids). The library population is depleted in Synthetic Helper Gene Product variants (or corresponding DNA barcodes) that reduce AAV titer. A portion of the AAV library was non- infectious (gray capsids).

[0060] Fig. 1 Part D. The packaged Synthetic Helper Gene Product library was transduced into fresh packaging cells that have all necessary components for viral production (e.g. transfected with packaging plasmids, stable genome integration of packaging plasmids, or helper virus). This transduction step selected for DNA sequences that encode Synthetic Helper Gene Products whose presence in the previous viral packaging step improved the production of viral particles harboring their own Synthetic Helper Gene Product DNA coding sequence.

Thus, Synthetic Helper Genes that express Synthetic Helper Gene Products that improved viral packaging gained a selective advantage that allows for increased nucleic acid transduction of such Synthetic Helper Genes into fresh packaging cells. This coupled the physiological impact of each Synthetic Helper Gene Product on viral packaging to the Synthetic Helper Gene sequence’s ability to propagate, establishing a strong selective pressure for Synthetic Helper Genes that enhanced viral titer or function/infectivity.

[0061] Fig. 1 Part E. Packaging cells (containing necessary elements for viral production) that are successfully transduced by a viral particle harboring a Synthetic Helper Gene produced a subsequent generation of viral particles harboring Synthetic Helper Genes. [0062] Fig. 1 Part F. The resulting library of Synthetic Helper Gene harboring viral particles were enriched for coding sequences for Synthetic Helper Gene Products that either increased physical titer or infectiousness (e.g., black particles) and depleted for coding sequences of Synthetic Helper Gene Products that interfere with viral packaging. At this point, there were more black, functional particles compared to gray, non-functional particles because of the selective enrichment for functional particles harboring Synthetic Helper Genes that enhance viral production.

[0063] Fig. 1 Part G. Subsequent rounds of enrichment are possible in a cyclic fashion by transducing the previous generation AAV-vectored Synthetic Helper Gene library into fresh packaging cells. As in D-F, the Synthetic Helper Genes that mediated production of AAVs with increased infectivity and/or increased titer are selected for. More infectious AAVs more efficiently delivered their DNA encoding the Synthetic Helper Gene Product into packaging cells, which results in a greater number of more infectious AAVs being produced. Less infectious AAVs delivered their DNA encoding the Synthetic Helper Gene Product into packaging cells less efficiently and are outcompeted. The coding sequences of Synthetic Helper Gene Products that mediate higher viral production titer and/or function had a similar selective advantage. This enrichment loop is repeated as many times as desired. 2-4 rounds of enrichment were observed to be optimal.

[0064] Fig. 1 Part H. After a desired number of rounds of selection, viral DNA is purified. DNA was purified at each round for NGS as described in Fig. 2 and Fig. 3 below. The ITRs are shown on either side of the gene as rectangular loops.

[0065] Fig. 1 Part I. The Synthetic Helper Gene library was moved from a viral transfer vector (e.g. that would be packaged into a viral particle as a result of ITRs) to a standard expression plasmid with no packaging sequences (i.e. so that it won’t be packaged into a viral particle). The resulting library was cloned using standard molecular biology techniques (e.g. transform into E. coli, grow on petri dishes, pick individual colonies into liquid media, purify DNA). The Synthetic Helper Gene library is now separated into many individual plasmid isolates, providing a convenient form for genotypic and phenotypic analysis. The circles are the plasmids. DNA sequencing is used to validate proper plasmid assembly and determine the Synthetic Helper Gene identity.

[0066] Fig. 1 Part J. The impact of individual Synthetic Helper Gene Products (and/or defined combinations) was evaluated by adding the Synthetic Helper Gene plasmids to a fresh viral packaging process.

[0067] Fig. 1 Part K. The resulting viral material produced by different Synthetic Helper Gene Product compositions was characterized (e.g. physical titer, biological titer, TU:VG ratio). Different Synthetic Helper Gene Product plasmids resulted in different viral titers (physical and biological) as well as infectivity metrics (TU:VG ratio) as depicted by different numbers and ratios of black, gray, and total viral particles. A cartoon of a bar chart represents data resulting from whatever form of analysis was performed.

[0068] Fig. 2. Exemplary discovery of Synthetic Helper Gene Products that enhance viral production by sequence analysis of viral genomes. [0069] Part A. A plasmid library of Synthetic Helper Gene flanked by two ITRs (SEQ ID NO: 8-9). The library may also include additional elements: fluorescent proteins (GFP), reporter enzymes (luciferase) or a barcode.

[0070] Fig. 2 Part B. Packaging cells were transfected with a library of genes configured to produce Synthetic Helper Gene Products from A. The Synthetic Helper Gene library and genes required for AAV production are expressed in host cells. Synthetic Helper Gene Products that interfere with AAV production also interfere with their own coding DNA being packaged into AAV. Synthetic Helper Gene Products that enhance AAV production will enhance the packaging of their own coding DNA into AAV particles.

[0071] Fig. 2 Part C. The Synthetic Helper Gene Product AAV library was harvested from the packaging cells. The AAV library harbors DNA encoding Synthetic Helper Gene Products or DNA barcodes that can be used to identify the Synthetic Helper Gene present inside of the cell that the AAV was produced in. The library population was enriched for Synthetic Helper Gene Product variants (or corresponding DNA barcodes) that enhanced AAV titer (black AAV capsids). The library population is depleted in Synthetic Helper Gene variants (or corresponding DNA barcodes) whose gene products reduced AAV titer. A portion of the AAV library is non-infectious (gray capsids).

[0072] Fig. 2 Part D. Cells (optionally configured for viral production) were transduced with the viral composition from Part C. Some of these viral particles will transduce either the Synthetic Helper Gene or a barcode that can be used to identify the Synthetic Helper Gene from A. The cells can optionally be in a living animal (e.g. mouse, primate), providing a means of evaluating the in vivo performance of viral material generated in the presence of Synthetic Helper Gene Products. Synthetic Helper Gene Products that, during packaging, increased either the viral titer or infectivity of the viral particles harboring their own coding DNA will have a selective advantage. This couples the impact of Synthetic Helper Gene Products on viral packaging to the Synthetic Helper Gene coding DNA’s ability to transduce its payload (i.e. itself), creating a facile means of quantitatively measuring (F) the impact of Synthetic Helper Gene Products on viral titer and infectivity by sequencing the Synthetic Helper Gene library DNA at different points.

[0073] Fig. 2 Part E. Viral genome DNA was harvested from the transduced cells in D (or from viral particles if cells in D were configured to produce a new generation of virus). The ITRs are shown on either side of the gene as rectangular loops.

[0074] Fig. 2 Part F. The impact of each Synthetic Helper Gene Product library member on viral production and performance was assayed by NGS. Naive Synthetic Helper Gene library DNA and DNA from AAVs that have been packaged and/or transduced are sequenced by NGS. AAVs harboring DNA that encodes Synthetic Helper Gene Products that alter AAV production (e.g. viral titer, infectiousness) were easily identified by comparing changes in relative population frequency (e.g. naive Synthetic Helper Gene library, packaged Synthetic Helper Gene library, re-packaged Synthetic Helper Gene library, transduced Synthetic Helper Gene library, etc. . .).

[0075] Fig. 2 Part G. This sequence-based approach for evaluating the impact of synthetic proteins on viral replication by using a functional virology system provided a novel, low cost, high throughput, facile, unbiased, and quantitative readout of the impact of a particular Synthetic Helper Gene Product on AAV production. Many Synthetic Helper Genes with desirable properties were rapidly identified. Desired Synthetic Helper Genes identified were subsequently evaluated for their impact on viral production in isolation and in different experimental contexts (e.g. in vivo, different media, different cell lines, etc. . .).

[0076] Fig. 2 Part H. Candidate Synthetic Helper Gene Products were added to cells configured to produce viral material (e.g. all necessary rep, cap, helper, transgenes).

[0077] Fig. 2 Part I. Viral material produced in the presence of Synthetic Helper Gene Products with high enrichment scores (e.g., increases in population from naive library to packaged library, from packaged library to transduced DNA sequence, from round to round, etc. . .) was characterized by measuring physical titer, biological titer, and/or potency (i.e. TU:VG ratio) in order to validate the ability of Synthetic Helper Gene Products to increase viral production.

[001] Fig. 3. Exemplary in vivo discovery of Synthetic Helper Gene Products that enhance viral production. This figure shows exemplary in vivo discovery of Synthetic Helper Gene Products that enhance viral production by sequencing transduced viral genomes encoding or associated with Synthetic Helper Gene Products.

[002] Fig. 3 Part A. Wildtype (WT) mammalian cells are transfected with DNA encoding Rep, Cap, helper genes and a library of Synthetic Helper Genes configured to produce Synthetic Helper Gene Products. The Synthetic Helper Genes, or a DNA barcode sequence allowing identification of the Synthetic Helper Gene variant, is flanked by two ITRs (SEQ ID NO: 8-9). The library may also include additional elements: fluorescent proteins (GFP), reporter enzymes (luciferase).

[003] Fig. 3 Part B. The Synthetic Helper Gene library and genes required for AAV production are expressed in host cells to produce a library of AAVs comprising Synthetic Helper Gene DNA encoding a Synthetic Helper Gene Product, or a DNA barcode sequence allowing identification of the Synthetic Helper Gene variant.

[004] Fig. 3 Part C. The AAV library is harvested. (The AAV library may optionally be enriched as described in Fig. 1G or Fig. 3 J.)

[005] Fig. 3 Part D. The AAV library is used to transduce an animal. Consideration should be given to whether expression of the Synthetic Helper Gene Product in the mouse is desirable. In some contexts, this may be desirable (e.g., for drug discovery). However, in many contexts, expression is not desirable and either the use of barcodes or inducible expression systems that prevent expression of the Synthetic Helper Gene Product in the animal model are preferred. [006] Fig. 3 Part E. Cells are harvested from the animal that was transduced by the AAV library.

[007] Fig. 3 Part F. DNA is harvested from the cells of the animal that were transduced by the AAV library and prepared for NGS.

[008] Fig. 3 Part G. DNA encoding Synthetic Helper Gene Products or barcodes identifying said Synthetic Helper Gene Products are sequenced by NGS. DNA from the naive library (Part A), the previous enrichment round, or the AAV-packaged library (Part C), as well as the in vivo transduction, are sequenced.

[009] Fig. 3 Part H. The relative impact of a Synthetic Helper Gene Product on AAV properties in vivo is determined by comparing the DNA sequence counts of Synthetic Helper Gene DNA from the in vivo selection to DNA from either the naive library (shown in A), previous enrichment rounds, or the AAV-packaged library (shown in C). AAVs that are more infectious in vivo will more efficiently deliver their Synthetic Helper Gene coding sequences or associated barcodes into cells/tissues/organs of the animal. Poorly functional or nonfunctional AAVs are cleared by the animal immune system. The cells/tissues/organs of the animal will be enriched in Synthetic Helper Gene DNA that encodes Synthetic Helper Gene Products (or associated barcode) conferring a selective/Darwinian advantage in infectivity, durability or other properties.

[0010] Fig. 4. Exemplary pathways for discovering Synthetic Helper Gene Products that enhance viral production encompassing functional enrichment and sequence-guided characterization of packaged or transduced viral genomes.

[0011] Fig. 4 Part A. Packaging cells were transfected with a library of genes configured to produce Synthetic Helper Gene Products and flanked by two ITRs (SEQ ID NO: 8-9). The library may also include additional elements: fluorescent proteins (GFP), reporter enzymes (luciferase) or a barcode. The packaging cells contained DNA encoding Rep (SEQ ID NO: 1-4, 10, 12, 14), Cap (SEQ ID NO: 5-7, 11, 13, 15), and helper genes (SEQ ID NO: 16- 23) (e.g. from triple transfection, stable integration, etc. . .).

[0012] Fig. 4 Part B. The Synthetic Helper Gene Product library and genes required for AAV production are expressed in host cells. Synthetic Helper Gene Products that reduce AAV production reduce their own coding DNA from being packaged. Synthetic Helper Gene Products that enhance AAV production enhanced the packaging of their own coding DNA into AAV virions.

[0013] Fig. 4 Part C. The library of AAV virions is harvested from the AAV packaging cells. The AAV library harbors DNA encoding Synthetic Helper Gene Products or DNA barcodes that can be used to identify the Synthetic Helper Gene Product present inside of the cell that the AAV was produced in. The library population is enriched for Synthetic Helper Gene Product variants (or corresponding DNA barcodes) that enhance AAV production (black AAV capsids). The library population is depleted in Synthetic Helper Gene variants (or corresponding DNA barcodes) that reduce AAV production (gray capsids).

[0014] Fig. 4 Part D. Multiple rounds of enrichment are possible in a cyclic fashion by transducing the previous generation of AAV Synthetic Helper Gene library into fresh packaging cells. Synthetic Helper Gene Products that mediate production of AAVs with increased infectivity and/or increased titer are selected for.

[0015] Fig. 4 Part E. Cells were transduced with the viral composition from C or D. These viral particles will transduce either the Synthetic Helper Gene(e.g. from D) or a barcode that can be used to identify the Synthetic Helper Gene from A (most suitable for in vivo experiments). The cells can optionally comprise a living animal (e.g. a mouse), providing a means of evaluating the in vivo impact of Synthetic Helper Gene Product on viral production. (See Fig. 3D for additional considerations.)

[0016] Fig. 4 Part F. Viral DNA that encodes Synthetic Helper Gene Products (or allows for their identification) was sequenced. Viral DNA from different rounds was sequenced to provide insight into the enrichment dynamics of Synthetic Helper Gene Products. [0017] Fig. 4 Part G. Sequence information from different rounds was used to determine the impact that various Synthetic Helper Gene Products have on viral packaging. For example, an increase in representation of a Synthetic Helper Gene in round 1 (e.g. naive plasmid library to first packaged library) is likely to indicate phenotypes that yield higher physical titer because there has been no selective pressure for increased infectivity. In contrast, Synthetic Helper Genes that showed negligible population increase in round 1 but display a noticeable increase in round 2 are more likely to mediate improvements in the production of infectious viral particles.

[0018] Fig. 4 Part H. The sequence-based analysis makes it easy to identify Synthetic Helper Gene Products that are useful for optimizing viral manufacturing.

[0019] Fig. 4 Part I. Synthetic Helper Genes can be used and characterized in a variety of ways (e.g. plasmid expressed Synthetic Helper Gene Products, genome expressed stable cell lines, helper virus borne, etc. . .).

[0020] Fig. 4 Part J. Viral production titer and/or potency was substantially increased when a Synthetic Helper Gene Product is present in the packaging cell.

[0021] Fig. 5. Exemplary molecular genetic flow of events that take place during Synthetic Helper Gene Product (SHGP)-modulated viral packaging, and a DNA sequence encoding a SHGP is packaged into a viral particle.

[0022] Fig. 5 Part A. Wild-type (WT) mammalian cells are transfected with DNA encoding Rep (SEQ ID NO: 1-4, 10, 12, 14), Cap (SEQ ID NO: 5-7, 11, 13, 15), and helper genes (SEQ ID NO: 16-23); as well as a Synthetic Helper Gene configured to produce a Synthetic Helper Gene Product and flanked by two ITRs. The payload DNA may also include additional DNA payload elements: fluorescent proteins (GFP), reporter enzymes (luciferase) or a barcode.

[0023] Fig. 5 Part B. A close-up of a cell produced in Part A.

[0024] Fig. 5 Part C. The Synthetic Helper Gene is expressed in the host cell while

AAV biosynthesis and assembly occur (see Part F).

[0025] Fig. 5 Part D. The transcribed and translated Synthetic Helper Gene Product.

[0026] Fig. 5 Part F. Synthetic Helper Gene Products perturb various aspects of host cell physiology, viral biology, or both. The exact target or mechanism is not required. These perturbations can either enhance (arrow) or inhibit (T-bar) biological processes involved in viral production.

[0027] Fig. 5 Part G. Synthetic Helper Gene DNA encoding Synthetic Helper Gene Product is configured to be packaged by AAV via flanking ITRs (SEQ ID NO: 8-9). In this way, Synthetic Helper Gene Products that enhance AAV production will enhance the packaging of their own Synthetic Helper Gene DNA (cDNA) into AAV virions. Synthetic Helper Gene Products that reduce cell viability or AAV assembly reduce their own coding DNA from being packaged. In this way, a Synthetic Helper Gene Product’s ability to enhance a packaging cell’s ability to produce AAV particles can be connected to its ability to replicate, providing for a powerful genetic selection, which allows for the rapid identification of Synthetic Helper Gene Products that enhance viral titer.

[0028] Fig. 6. Exemplary molecular genetic flow of events that take place during SHGP-modulated viral packaging, where SHGP is provided on a plasmid, a DNA barcode sequence is packaged into a viral particle, but the SHGP is NOT packaged into a viral particle. [0029] Fig. 6 Part A. WT Mammalian cells are transfected with DNA encoding Rep, Cap, and helper genes; a Synthetic Helper Gene configured to produce Synthetic Helper Gene Products in such a way that it will not be packaged by AAV; as well as DNA barcode to identify the Synthetic Helper Gene Product that is flanked by two ITRs (SEQ ID NO: 8-9). The Payload DNA may also include additional DNA payload elements: fluorescent proteins (GFP), reporter enzymes (luciferase).

[0030] Fig. 6 Part B. A close-up of a cell produced in Part A.

[0031] Fig. 6 Part C. The Synthetic Helper Gene is expressed in the host cell while

AAV biosynthesis and assembly occur (see Part F).

[0032] Fig. 6 Part D. The transcribed and translated Synthetic Helper Gene Product.

[0033] Fig. 6 Part F. Synthetic Helper Gene Products perturb various aspects of host cell physiology, viral biology, or both. The exact target or mechanism is not required. These perturbations can either enhance (arrow) or inhibit (T-bar) biological processes involved in viral production.

[0034] Fig. 6 Part G. Synthetic Helper Gene DNA is operably linked to a DNA barcode flanked by two ITRs (SEQ ID NO: 8-9). The DNA barcode is configured to be packaged by AAV via flanking ITRs (SEQ ID NO: 8-9), but the Synthetic Helper Gene is configured not to be packaged. In this way, Synthetic Helper Gene Products that enhance AAV production will enhance the packaging of a DNA barcode used to identify the Synthetic Helper Gene Product into AAV virions. Synthetic Helper Gene Products that reduce cell viability or AAV assembly reduce the packaging of their identifying DNA barcode from being packaged. In this way, a Synthetic Helper Gene Product’s ability to enhance a AAV production in a packaging cell can be connected to its identity (via the barcode), providing for a powerful genetic selection, which allows for the rapid identification of viral production-enhancing Synthetic Helper Gene Products.

[0035] Fig. 7. Exemplary molecular genetic flow of events that take place during Synthetic Helper Gene Product-modulated viral packaging, using a quadruple transfection in which Synthetic Helper Gene Product is supplied on plasmid and not packaged, while the pAAV payload is packaged into a viral particle. [0036] Fig. 7 Part A. WT Mammalian cells are transfected with DNA encoding Rep, Cap, and helper genes; as well as a gene configured to produce Synthetic Helper Gene Products; and a DNA payload flanked by two ITRs (SEQ ID NO: 8-9) that is to be packaged into the AAV virion.

[0037] Fig. 7 Part B. A close-up of a cell produced in Part A.

[0038] Fig. 7 Part C. The Synthetic Helper Gene Product is expressed in the host cell while AAV biosynthesis and assembly occur (see part E).

[0039] Fig. 7 Part D. The transcribed and translated Synthetic Helper Gene Product.

[0040] Fig. 7 Part E. Synthetic Helper Gene Products perturb various aspects of host cell physiology, viral biology, or both. The exact target or mechanism is not required.

Perturbations that enhance (arrow) biological processes involved in viral production increase packaging of the ITR-flanked DNA payload into AAV virions.

[0041] Fig. 7 Part F. The ITR-flanked DNA payload is packaged into an AAV virion. In contrast, the Synthetic Helper Gene is not packaged into the virion. This approach was used to characterize the effects of individual Synthetic Helper Gene Products on AAV production (e.g. increased physical titer, biological titer, transduction efficiency, etc. . .).

[0042] Fig. 8. Exemplary molecular genetic flow of events that take place during SHGP -modulated viral packaging, where Synthetic Helper Gene Product is supplied by expression from a stable genome integration and a pAAV payload is packaged into a viral particle.

[0043] Fig. 8 Part A. WT Mammalian cells are transfected with DNA encoding Rep, Cap, and helper genes; as well as a DNA payload flanked by two ITRs (SEQ ID NO: 8-9) that is to be packaged into the AAV virion. A Synthetic Helper Gene expression cassette is genomically integrated into the host cell.

[0044] Fig. 8 Part B. A close-up of a cell produced in Part A.

[0045] Fig. 8 Part C. The Synthetic Helper Gene is expressed in the host cell while

AAV biosynthesis and assembly occur (see part E).

[0046] Fig. 8 Part D. The transcribed and translated Synthetic Helper Gene Product.

[0047] Fig. 8 Part E. Synthetic Helper Gene Products perturb various aspects of host cell physiology, viral biology, or both. The exact target or mechanism is not required.

Perturbations that enhance (arrow) biological processes involved in viral production increase packaging of the ITR-flanked DNA payload into AAV virions. [0048] Fig. 8 Part F. The ITR-flanked DNA payload is packaged into an AAV virion. In contrast, the Synthetic Helper Gene is not packaged into the virion because it is located on the host cell’s chromosome.

[0049] Fig. 9. Exemplary basis for enrichment of Synthetic Helper Gene Products that enhance viral packaging (left panel) and depletion of Synthetic Helper Gene Products that disrupt viral packaging (right panel).

[0050] Fig. 9 Part A. A cell synthetic to produce AAV with a Synthetic Helper Gene Product that improves AAV production.

[0051] Fig. 9 Part B. An ITR-flanked Synthetic Helper Gene Product expression cassette is transcribed and translated.

[0052] Fig. 9 Part C. Synthetic Helper Gene Products perturb various aspects of host cell and/or viral physiology. The exact target or mechanism is not required.

[0053] Fig. 9 Part D. Because this Synthetic Helper Gene Product enhances AAV production, packaging of Synthetic Helper Gene DNA into AAV virions is increased.

[0054] Fig. 9 Part E. A cell synthetic to produce AAV with a Synthetic Helper Gene Product that reduces AAV production.

[0055] Fig. 9 Part F. An ITR-flanked Synthetic Helper Gene expression cassette is transcribed and translated.

[0056] Fig. 9 Part G. Synthetic Helper Gene Products perturb various aspects of host cell and/or viral physiology. The exact target or mechanism is not required.

[0057] Fig. 9 Part H. Because this Synthetic Helper Gene Product is toxic or otherwise interferes with processes involved in AAV production, packaging of Synthetic Helper Gene DNA into AAV virions is reduced.

[0058] Fig. 10. Exemplary basis for enrichment of Synthetic Helper Gene Products that increase viral titer and/or transduction efficiency of infectious AAV particles.

[0059] Fig. 10 Part A. WT Mammalian cells are transfected with DNA encoding Rep, Cap, and helper genes. The same WT mammalian cells are also transduced by AAV harboring a Synthetic Helper Gene configured to produce a Synthetic Helper Gene Product. The input AAV may, for example, be the result of a previous round of Synthetic Helper Gene enrichment.

[0060] Fig. 10 Part B. A close-up of a cell produced in Part A.

[0061] Fig. 10 Part C. Successful AAV transduction of the Synthetic Helper Gene

DNA requires the AAV particle to bind the host cell, enter the cell, navigate cell trafficking, escape the endosome, avoid proteasomal destruction, enter the nucleus, uncoat, release DNA, process DNA, and avoid innate antiviral immune response. Successful transduction results in the AAV DNA payload containing the Synthetic Helper Gene release and expression in the host cell.

[0062] Fig. 10 Part D. Successfully delivered Synthetic Helper Genes are transcribed and translated by the host cell machinery.

[0063] Fig. 10 Part E. Synthetic Helper Gene Product modulates host cell and/or viral physiology during AAV production to generate a subsequent generation of AAV particles with increased infectiousness. The exact target or mechanism is not required (see part C. for examples).

[0064] Fig. 10 Part F. Synthetic Helper Gene DNA sequences that more efficiently transduce cells and express Synthetic Helper Gene Products that improve the transduced cell’s ability to produce more infectious AAV will enhance the packaging of their own coding DNA into more infectious AAV virions. This confers a replicative advantage to DNAs encoding Synthetic Helper Gene Products the optimize host cells for AAV production.

[0065] Fig. 11. Exemplary basis for selective enrichment of Synthetic Helper Gene sequences that improve viral production and depletion of Synthetic Helper Gene sequences that reduce viral production. This Figure provides an example of the dynamics at play during selection of Synthetic Helper Gene Products that enhance viral packaging. Synthetic Helper Gene Products that generate more infectious AAVs can more efficiently get their DNA encoding the Synthetic Helper Gene Product into packaging cells, which results in a greater number of more infectious AAVs being produced (possibly akin to a “K-selected” reproductive strategy). Similarly, Synthetic Helper Gene Products that generate greater numbers of infectious SHG-AAVs have more chances to transduce their DNA into packaging cells, resulting in ever greater numbers of AAVs harboring these Synthetic Helper Gene being produced (possibly akin to a “R-selected” reproductive strategy).

[0066] Fig. 11 Part A. Cells with DNA encoding Rep, Cap, helper genes; as well as pSHG, an ITR-flanked gene configured to produce Synthetic Helper Gene Products that increase AAV production.

[0067] Fig. 11 Part B. AAV virions produced in cells expressing Synthetic Helper Gene Products that improved AAV viral titer and/or transduction efficiency harbor the Synthetic Helper Gene sequences that improved those viral production attributes. These AAV particles infect other cells configured to produce AAVs (e.g., neighboring cells in the same round of enrichment or fresh cells in a subsequent round of enrichment). [0068] Fig. 11 Part C. AAV borne Synthetic Helper GeneS that enhance viral production are amplified through multiple transductions and AAV replication cycles and can rapidly outcompete Synthetic Helper GeneS conferring modest to negative effects on viral packaging (Parts D-E). Thus, Synthetic Helper Gene sequences that improve viral production are rapidly enriched.

[0069] Fig. 11 Part D. Cells with DNA encoding Rep, Cap, helper genes; as well as pSHG, an ITR flanked gene configured to produce Synthetic Helper Gene Products that interfere with AAV production.

[0070] Fig. 11 Part E. Cells expressing Synthetic Helper Gene Products that interfere with AAV production do not generate AAV particles harboring these Synthetic Helper Gene constructs at sufficient quantities or levels of infectiousness to effectively compete against Synthetic Helper Gene Products that improve viral production. Thus, Synthetic Helper Gene sequences that reduce viral production are rapidly depleted.

[0071] Fig. 11 Part F. Viral genomes can be collected and analyzed through NGS to characterize the effect of specific Synthetic Helper Gene Products on the production of AAV particles.

[0072] Fig. 12. Exemplary Basis for analysis of Synthetic Helper Gene Products (SHGPs) that improve or decrease viral production performance in cells not configured to produce additional AAV particles.

[0073] Fig. 12 Part A. Cells with DNA encoding Rep, Cap, helper genes; as well as pSHG, an ITR flanked gene configured to produce Synthetic Helper Gene Products that increase (top) or decrease (bottom) AAV production.

[0074] Fig. 12 Part B. Cells infected by AAV particles that were produced in a cell in the presence of a Synthetic Helper Gene Product that increased AAV production are able to deliver their DNA payload (a Synthetic Helper Gene or associated barcode) more efficiently or in greater numbers compared to AAV particles produced in the presence of Synthetic Helper Gene Products that reduce AAV production.

[0075] Fig. 12 Part C. Viral genomes can be collected and analyzed through NGS to characterize the effect of specific Synthetic Helper Gene Products on the production of AAV particles.

[0076] Fig. 13. Exemplary molecular genetic flow of events that take place during SHGP -modulated packaging of lentiviral vectors. [0077] Fig. 13 Part A. A plasmid borne Synthetic Helper Gene can be used to generate Synthetic Helper Gene Products during lentiviral packaging. This plasmid is co-transfected with the rest of the packaging plasmids (see B and C).

[0078] Fig. 13 Part B. The Lentiviral packaging DNA provides the necessary components for viral production. There are a variety of different systems; this figure illustrates a typical “3rd generation” system. This system splits the viral genes across multiple plasmids to reduce the risk of generating replication competent viral particles. Typically, a third- generation lentiviral packaging system consists of four plasmids, the gag-pol, env, rev, and transfer plasmid (C). Env, the envelope plasmid, expresses the viral envelope glycoprotein (SEQ ID NO: 37). Gag-pol expresses structural proteins (Gag) (SEQ ID NO: 26) and enzymes (Pol) (SEQ ID NO: 25). The Gag protein includes matrix (MA), capsid (CA), and nucleocapsid proteins (NC). Pol includes reverse transcriptase, integrase, and protease (pro). Rev (SEQ ID NO: 24) expresses the Rev protein which facilitates nuclear export of the unspliced or partially spliced viral RNAs.

[0079] Fig. 13 Part C. pTrans, (the transfer plasmid) contains the payload transgene of interest (e.g. a therapeutic payload) along with the long terminal repeats (LTRs) (SEQ ID NO: 28-29), the Psi packaging signal (SEQ ID NO: 34)), and typically the Rev response element (RRE). Some systems also include a central polypurine tract (cPPT) to enhance nuclear import of the pre-integration complex.

[0080] Fig. 13 Part D. A close up of a cell when all 5 components (the SHGP plasmid and the packaging plasmids / transfer vector) are transfected into packaging cells (e.g., HEK293 cells). These cells begin to produce lentiviral particles under the influence of Synthetic Helper Gene Products.

[0081] Fig. 13 Part E. The Synthetic Helper Gene is transcribed and translated.

[0082] Fig. 13 Part F. The lentiviral packaging DNA is expressed, allowing the cell to generate viral particles.

[0083] Fig. 13 Part G. The Synthetic Helper Gene Product modulates cellular environment in order to enhance viral production.

[0084] Fig. 13 Part H. The transcribed RNA transgene is packaged into a lentivirus.

[0085] Fig. 13 Part I. The transgene is packaged into a lentivirus, but the SHGP sequence is not.

[0086] Fig. 14. Exemplary architectures of antibody-like Synthetic Helper Gene libraries. [0087] Fig. 14 Part A. Exemplary architecture of a Synthetic Helper Gene encoded inside two functional ITRs (SEQ ID NO: 8-9) on an AAV transfer vector. In this case, a nanobody is used (exemplary SEQ ID NOs: 215-216) . The library may also include additional elements: fluorescent proteins (such as green fluorescent protein or GFP), reporter enzymes (luciferase) or a barcode.

[0088] Fig. 14 Part B. A library is created by inserting degenerate codons, depicted as “NNK”, into the variable region of the nanobody; this region determines the specificity of nanobody binding.

[0089] Fig. 14 Part C. Complementarity-determining region 3 (CDR3) of the nanobody is the most variable portion of the nanobody. CDR3 recognizes and binds specific epitopes. [0090] Fig. 14 Part D. A plasmid used to transfect packaging cells (not shown) with a library of genes configured to produce antibody-like Synthetic Helper Genes based on nanobody scaffolds. The packaging cells contain DNA encoding Rep, Cap, and helper genes (e.g. from triple transfection, stable integration, etc.) required for AAV production.

[0091] Fig. 14 Part E. The Synthetic Helper Genes are expressed in host cells and generate antibody-like SHGPs, represented as a circle.

[0092] Fig. 14 Part F. The Synthetic Helper Gene Products that reduce AAV production reduce their own coding DNA from being packaged. Synthetic Helper Gene Products that enhance AAV production will enhance the production of their own coding DNA into AAV virions.

[0093] Fig. 14 Part G. In an alternate architecture, the Synthetic Helper Gene can be encoded outside the ITRs (SEQ ID NO: 8-9). A unique DNA barcode sequence is instead included inside the ITRs (SEQ ID NO: 8-9). The DNA barcode is packaged during AAV packaging while the Synthetic Helper Gene is not.

[0094] Fig. 15. Exemplary architectures of engineered transcription factor Synthetic Helper Gene libraries generated with error prone PCR.

[0095] Fig. 15 Part A. Overall structure of the SHG encoded within two functional ITRs (e.g. SEQ ID NO: 8-9) on an AAV transfer vector and an optional payload (e.g. GFP, therapeutic payloads). This is the generic structure employed in the development of SEQ ID NOs: 115 - 214.

[0096] Fig. 15 Part B. The library is created by introducing mutations into the SHG DNA sequence through error-prone PCR, represented as "Mutagenized SHG DNA".

[0097] Fig. 15 Part C. The mutagenized SHG DNA is inserted into the transfer vector so that it is operably linked to ITRs. [0098] Fig. 15 Part D. Packaging cells are transfected with the library of mutagenized SHG genes. These cells contain the necessary components for AAV production.

[0099] Fig. 15 Part E. The mutagenized SHG is expressed in host cells, producing mRNA, which is then translated into the SHG (circle).

[00100] Fig. 15 Part F. The expressed SHG products influence AAV production. Those enhancing production will increase their own packaging into AAV virions, while those reducing production will decrease their packaging.

[00101] Fig. 15 Part G. An alternative architecture where the mutagenized SHG is encoded outside the ITRs. A DNA barcode is included within the ITRs for packaged identification.

[00102] Fig. 16. Exemplary volcano plot illustrating Synthetic Helper Gene Protein enrichment, depletion, confidence, and NGS Read count.

[00103] This figure presents a volcano plot of SHGP library enrichment in AAV packaging cells. This particular plot is an exemplary result from a round 2 enrichment on the mutagenized NLH Nanobody hPEST T2A eGFP in Takara pCMV - with saturation mutagenized CDR3. Similar plots were generated for other SHGPs and rounds of selection, but are not shown for brevity.

[00104] Values on the positive x-axis signify nanobodies that were positively enriched, indicating increased fitness during viral packaging-the desired phenotype. The y-axis reflects the confidence in these observations in -loglO(p-val). Each point represents data for a single Nanobody sequence. The size of the point corresponds to the read depth observed in NGS (sum of naive & enriched). Points in gray are at or below the limit of detection (i.e. one or less counts detected in NGS). The two horizontal dashed lines represent loglO (enrichment=10). The single horizontal dashed line represents -loglO (P-value=0.05); anything above this is considered statistically significant.

[00105] Nanobody sequences in the upper right corner are both highly enriched and the most statistically significant. As can be observed in the figure, a significant number of Nanobody appear to be strongly enriched. This proves that there are many Nanobody that can be used to substantially increase AAV packaging performance. The plot also allows for the identification of potentially detrimental SHGPs in the upper left quadrant, if any, which might impair AAV packaging or be toxic to the cells.

[00106] To provide additional insight into the unexpected scale and efficiency with respect to identifying Synthetic Helper Gene Proteins that improve viral yield, more than 62,409 unique Synthetic Helper Gene Protein Products were observed to be enriched by at least 10-fold (X-axis>l) with a P-value of less than 0.05 (Y-axis>~1.3 = -logl0(0.05)). SEQ ID NOs: 55 - 74 were identified in this library and they are representative members.

[00107] Fig. 17. Exemplary T-SNE sequence clustering of some Synthetic Helper Gene Protein library diversity.

[00108] Overview: t-SNE scatter plots illustrating clusters of similar Synthetic Helper Gene Proteins based on NLH (black points in A, light points in B) or Zimmermann (light points in A, black points in B) Nanobodies. T-SNE (t-distributed Stochastic Neighbor Embedding) is a dimensionality reduction technique used to visualize high-dimensional data in a 2D or 3D space. In this plot, the x and y axes do not represent specific features but rather arbitrary dimensions that t-SNE creates to best preserve the relationships between data points in the original high-dimensional space. The closer two points are in this 2D representation, the more similar their features are in the original 768-dimensional space. It's important to note that the absolute positions on the x and y axes are not meaningful; only the relative distances between points matter. The 2D t-SNE representation was calculated on Synthetic Helper Gene Protein Product protein sequence embedding vectors of 768 dimensions generated using a pretrained protein sequence transformer encoder model ESM2. Each dot is an individual Synthetic Helper Gene Protein. Similar Synthetic Helper Gene Proteins (e.g., those with similar sequence, structural, or other features) cluster together, while dissimilar Synthetic Helper Gene Proteins are positioned further apart. We clustered the mutant libraries from two different nanobody parent scaffolds on the same plots in order to demonstrate that they would cluster distinctly as shown in the plots (black and gray do not overlap).

[00109] Fig. 17 Part A illustrates SHGPs identified from NLH Nanobody hPEST T2A eGFP in Takara pCMV parent scaffold with a mutagenized CDR3 following 1 round of enrichment. SEQ ID NOs: 35 - 54 were identified in 17A.

[00110] Fig. 17 Part B illustrates SHGPs identified from Zimmermann Nanobody hPEST T2A eGFP in Takara pCMV parent scaffold with a mutagenized CDR3 following 1 round of enrichment. SEQ ID NOs: 75 - 94 were identified in 17B.

[00111] This plot illustrates the breadth of sequence diversity that is reachable in just 2 SHG saturation mutagenesis libraries.

[00112] Fig. 18. Exemplary Amino Acid Frequency Plots for Zimmermann Nanobody hPEST T2A eGFP in Takara pCMV with saturation mutagenized CDR3.

[00113] Overview: Fig. 18 presents three amino acid frequency plots (a, b, c) for a mutant library of the Zimmermann Nanobody hPEST T2A eGFP construct in Takara pCMV, with saturation mutagenesis focusing on the CDR3 region. These plots are derived from Round 1 of selection. Each plot is a heatmap where the x-axis represents the amino acid sequence positions of an arbitrary reference protein (SEQ ID NO: 75 in this case), and the y-axis represents the 20 standard amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y). The grayscale intensity in each cell indicates the frequency of a specific amino acid at a particular position, with lighter shades representing higher frequencies and darker shades representing lower frequencies. This plot is conceptually similar to a deep mutational scanning plot, but is focused on amino acid frequencies across the entire library as opposed to the sequence function relationship of a particular protein sequence.

[00114] Fig. 18 Part A: This subplot displays the amino acid frequencies for variants with an enrichment score greater than 100. The heatmap shows distinct patterns of amino acid preferences across the sequence. Certain positions in CDR3 exhibit strong enrichment, indicated by the light pixels in the checkered portion of CDR3.

[00115] Fig. 18 Part B: This plot is essentially the same as Fig. 18 Part A, with the enrichment score threshold at 10 instead of 100. This lower threshold results in dramatically different amino acid frequencies at CDR3 as can be observed by a stark change in the checkering pattern. These differences highlight how the selection pressure changes as the enrichment score threshold is lowered.

[00116] Fig. 18 Part C: This plot is essentially the same as Fig. 18 Part A and 18 Part B, but now the enrichment threshold is set at less than 10 (i.e. 0.1 or lower or depleted by 10X or more). As expected, CDR3 amino acid frequencies show an obviously different pattern compared to CDR3s in Fig. 18 Part A and Fig. 18 Part B.

[00117] Conclusion: The amino acid frequency plots in Fig. 18 reveal clear and distinct patterns of amino acid preferences corresponding to different enrichment levels. There are noticeable changes in the amino acid frequency patterns of CDR3 depending on how the enrichment threshold is applied. This indicates how different mutations affect the nanobody's performance as a SHGP. These plots provide valuable insights into the sequence-function relationship of the Zimmermann Nanobody (and other nanobodies), offering guidance for further engineering efforts to optimize its properties. It also illustrates/teaches a facile means of rapidly specifying the sequence space of high performance SHGPs derived from other binder proteins.

[00118] Fig. 19. Exemplary distance matrix illustrating pairwise Euclidean distance of Synthetic Helper Gene Protein sequence embeddings.

[00119] This figure presents a distance matrix and accompanying dendrogram that visualize the sequence similarities among a select group of SHGPs. These proteins were derived from NLH Nanobody hPEST T2A eGFP in Takara pCMV with mutagenized CDR3 after one round of enrichment.

[00120] The main square grid displays pairwise distances between SHGP embeddings, with darker colors indicating smaller distances (higher similarity) and lighter colors representing greater distances (less similarity). The diagonal line appears darkest as it represents each sequence's comparison with itself, resulting in zero distance. Clusters of dark squares suggest groups of closely related sequences. The dendrogram on the left provides a hierarchical view of these relationships, with branches closer to the root (left side) indicating greater similarity between clusters.

[00121] To generate this visualization, we first employed a pre-trained protein sequence transformer encoder model (ESM2) to create 768-dimensional embedding vectors for each SHGP sequence. We then identified similar groups of sequences through k-means clustering. These clusters are approximately visualized separately in a t-SNE plot (Fig. 17 Part A). From each cluster (group of similar protein sequences), we selected the top-performing SHGP based on enrichment score. Thus, each protein sequence chosen for distance matrix analysis represents a high-performing sequence from a set of neighboring sequence variants.

[00122] The distance matrix was constructed by calculating the Euclidean distance between these embedding vectors for all pairs of selected sequences (i.e., the best sequence from each k-means cluster). Hierarchical clustering was applied to organize the sequences and generate the dendrogram, grouping similar sequences together and allowing for easy identification of related protein variants.

[00123] In the dendrogram, each horizontal line (leaf) corresponds to an individual SHGP sequence and aligns with a row in the distance matrix. The vertical lines (nodes) represent sequence clusters, with the height of each node indicating the degree of dissimilarity between the clusters it joins.

[00124] This analysis provides insights into the diversity and relationships among the most successful SHGP variants, helping to identify potential families of sequences with similar properties or functions.

[00125] Fig. 20. Exemplary t-SNE scatter plot illustrating sequence diversity and deep mutational scanning illustrating sequence-function relationships

[00126] Overview: Fig. 20 presents analysis of sequence diversity and sequencefunction relationships in a dinJ derived transcription factor based Synthetic Helper Gene Protein (SHGP) library. This figure combines two complementary visualization techniques to provide insights into both the overall sequence landscape and the functional impact of specific mutations.

[00127] Fig. 20 Part A presents a portion of a t-SNE plot. The Fig. 17 description provides the necessary information as the t-SNE plot presented here was generated identically, with the only difference being that the SHGP sequence library was dinJ. SEQ ID NOs: 115 - 134 we identified from this library.

[00128] Fig. 20 Part B presents a heatmap plot derived from Deep Mutational Scanning (DMS) on a Synthetic Helper Gene Protein (SHGP). DMS offers a comprehensive look at single-site amino acid substitutions in the SHGP, which are depicted visually on a heatmap. This depiction demonstrates the impact these mutations have on the protein's phenotypic properties (viral production in our case), as determined by NGS enrichment scores. The X-axis of the heatmap represents the amino acids found in the original SHGP, corresponding to SEQ ID NO: 115, while the Y-axis indicates the 20 natural amino acid substitutions at each residue position of the helper gene.

[00129] Each pixel in the heatmap corresponds to the enrichment of an SHGP when the amino acid residue, represented by a particular column, is mutated to the residue of a certain row. Enrichment is expressed through shades of gray: lighter colors suggest strong enrichment and thus beneficial mutations, while darker colors denote depletion, indicating disadvantageous mutations. A uniform gray suggests the mutant was either below the limit of detection or not present in the library. The brightest white pixels represent the amino acid of the protein sequence that is being analyzed because we chose the sequence for having the highest enrichment. However, the other enrichment factors observed in the single site mutations are still high, often exceeding order of magnitude.

[00130] This heatmap offers a comprehensive exploration of the SHGP's mutational landscape, revealing both existing and potential SHGP variants along with their performance characteristics. It provides valuable insights for protein engineering and molecular design by illustrating the functional consequences of amino acid substitutions. Patterns within the heatmap can identify crucial structural or functional regions, guiding optimization efforts. The plot also highlights gaps in mutational coverage, suggesting areas for further exploration. Leveraging this extensive sequence-function data, predictive models can be developed to prioritize variants beyond the original library.

[00131] Conclusion: Fig. 20 provides both a high-level view of the sequence landscape with a t-SNE plot (part A) and a more granular analysis of how specific mutations affect protein function with a DMS plot (part B). Together, this data illustrates methods for developing, understanding, and analyzing SHGPs. It provides quantitative insights into sequence diversity and sequence-function relationships that can be readily applied to other SHGP variants beyond those specifically described here.

[00132] Fig. 21. Exemplary t-SNE scatter plot illustrating sequence diversity and deep mutational scanning illustrating sequence-function relationships.

[00133] Fig. 21 follows the same structure as Fig. 20. Fig. 21 Part A corresponds to Fig.

20 Part A (refer to Fig. 17 for details), and Fig. 21 Part B corresponds to Fig. 20 Part B. The key differences are that Fig. 21 Part A depicts a mazE-derived SHGP library, which yielded SEQ ID NOs: 135-154 and Fig. 21B shows a DMS plot for SEQ ID NO: 135.

[00134] Fig. 22. Exemplary t-SNE scatter plot illustrating sequence diversity and deep mutational scanning illustrating sequence-function relationships.

[00135] Fig. 22 follows the same structure as Fig. 20. Fig. 22 Part A corresponds to Fig. 20 Part A (refer to Fig. 17 for details), and Fig. 22 Part B corresponds to Fig. 20 Part B. The key differences are that Fig. 22 Part A depicts a relB-derived SHGP library, which yielded SEQ ID NOs: 155-174 and Fig. 22 Part B shows a DMS plot for SEQ ID NO: 155.

[00136] Fig. 23. Exemplary t-SNE scatter plot illustrating sequence diversity and deep mutational scanning illustrating sequence-function relationships.

[00137] Fig. 23 follows the same structure as Fig. 20. Fig. 23 Part A corresponds to Fig.

20 Part A (refer to Fig. 17 for details), and Fig. 23 Part B corresponds to Fig. 20 Part B. The key differences are that Fig. 23 Part A depicts an rnlB-derived SHGP library, which yielded SEQ ID NOs: 175-194 and Fig. 23 Part B shows a DMS plot for SEQ ID NO: 175.

[00138] Fig. 24. Exemplary t-SNE scatter plot illustrating sequence diversity and deep mutational scanning illustrating sequence-function relationships.

[00139] Fig. 24 follows the same structure as Fig. 20. Fig. 24 Part A corresponds to Fig.

20 Part A (refer to Fig. 17 for details), and Fig. 24 Part B corresponds to Fig. 20 Part B. The key differences are that Fig. 24 Part A depicts a yefM-derived SHGP library, which yielded SEQ ID NOs: 195-214 and Fig. 24 Part B shows a DMS plot for SEQ ID NO: 195.

[00140] Fig. 25. Exemplary plots illustrating library diversity and coverage, enrichment profiles, and performance of individual sequences for an antibody-like SHGP based on a nanobody after one round of selection

[00141] Overview: This figure illustrates the diversity and enrichment profile of an exemplary antibody -like SHGP saturation mutagenesis library, based on the NLH Nanobody, after a single round of selection. [00142] Fig. 25 Part A.Count Data Histogram: This histogram depicts the distribution of antibody-like SHGP sequence counts obtained via next-generation sequencing (NGS). The X- axis represents the loglO of the number of times a specific sequence is observed, while the Y- axis shows the loglO frequency of sequences with that count. The distribution is heavily skewed towards low-count sequences (highest bars at left), indicating a library with high diversity and even distribution. Few sequences are over-represented, validating the library's quality. The data demonstrate a high-quality library with a vast diversity of sequences, with the majority occurring at low frequencies, as expected.

[00143] Fig. 25 Part B. Enrichment Data Histogram: This histogram shows the enrichment profile of the antibody-like SHGP library. The X-axis represents the loglO of the enrichment value for each sequence, calculated as the ratio of post-selection frequency to preselection frequency. The Y-axis shows the loglO frequency of sequences with that enrichment value. Notably, a significant portion of the library exhibits positive enrichment, indicating an unexpected prevalence of SHGPs that enhance viral production. This unexpected bias towards enriched sequences suggests that these SHGPs may optimize the host cell environment for viral packaging, a valuable and unanticipated finding. The selection process also amplifies high-performing sequences that may have been initially below detection limits, revealing their positive impact on viral production.

[00144] Fig. 25 Part C. This bar chart provides viral titer measurements for selected antibody-like SHGP sequences (SEQ ID NOs: 35-54) identified in the enrichment analysis (Fig. 25 Part B). The Y-axis shows logl0(VG/ml). The dark gray bar provides the reference viral titer, while the light gray bar provides the viral titer of the most enriched SHGP variants. A substantial increase in viral titer is apparent.

[00145] Conclusion: This figure demonstrates the utility of the described selection method in identifying novel antibody-like SHGPs with desirable properties from a highly diverse library. The unexpected finding of numerous SHGPs with increased viral production highlights the potential of this approach for generating antibody-like SHGPs that substantially increase viral production performance.

[00146] Fig. 26. Exemplary plots illustrating library diversity and coverage, enrichment profiles, and performance of individual sequences for an antibody-like SHGP based a Nanobody after two rounds of selection.

[00147] This figure follows the same format as Fig. 25, illustrating the diversity, enrichment, and performance of a saturation mutagenesis library for an antibody-like SHGP. For detailed explanations of Fig. 26 Parts A, B, and C, please refer to the description of Fig. 25. The sequences shown in Fig. 26 Part C correspond to SEQ ID NOs: 55-74.

[00148] Of note are the distinct bulges in the panel A histogram. The bulge in counts (Fig. 26 Part A) is a result of the high degree of enrichment in round 2. In other words, enough sequences were enriched in round 2 that the high-performing SHGPs can be directly visualized in the histogram. Of additional is that round 2 selections require that the AAV particles harboring the SHGPs be capable of transducing the payload into fresh packaging cells. This imposes a selective pressure for SHGPs that increase biological titer, typically measured in TU/ml. The enrichment histogram (Fig. 26 Part B) shows strong enrichment profiles for many SHGPs capable of transducing packaging cells. In the case of the top 20 SHGPs provided in part C, enrichment was negligible in the previous round 1, while the enrichment was much higher in round 2. This means that the round 2 enrichment successfully selected for SHGPs that increased biological titer. Thus, the observed increase in VG/ml can be approximated to an increase in TU/ml. Fig. 35 Parts A and B provide further evidence of increases in transduction efficiency.

[00149] Fig. 27. Exemplary plots illustrating library diversity and coverage, enrichment profiles, and performance of individual sequences for an antibody-like SHGP based on a Nanobody after one round of selection

[00150] This figure follows the same format as Fig. 25, illustrating the diversity, enrichment, and performance of a saturation mutagenesis library for an antibody-like SHGP. For detailed explanations of Fig. 27 Parts A, B, and C, please refer to the description of Fig. 25. The sequences shown in Fig. 27 Part C correspond to SEQ ID NOs: 75-94.

[00151] Fig. 28. Exemplary plots illustrating library diversity and coverage, enrichment profiles, and performance of individual sequences for an antibody-like SHGP based on a Nanobody after two rounds of selection

[00152] This figure follows the same format as Fig. 25, illustrating the diversity, enrichment, and performance of a saturation mutagenesis library for an antibody-like SHGP. For detailed explanations of Fig. 28 PartsA, B, and C, please refer to the description of Fig. 25. The sequences shown in Fig. 28 Part C correspond to SEQ ID NOs: 95-114.

[00153] As in Fig. 26, because this is a second-round library, the histograms in Fig. 28 Parts A and B have a more apparent bias resulting from the additional enrichment performed in the first round. [00154] Fig. 29. Exemplary plots illustrating library diversity and coverage, enrichment profiles, and performance of individual sequences for an engineered transcription factor SHGP based on dinJ.

[00155] This figure follows the same format as Fig. 25, illustrating the diversity, enrichment, and performance of an error-prone PCR library for an engineered transcription factor SHGP based on dinJ. For detailed explanations of Fig. 29 Parts A, B, and C, please refer to the description of Fig. 25. The sequences shown in Fig. 29 Part C correspond to SEQ ID NOs: 115-134.

[00156] Fig. 30. Exemplary plots illustrating library diversity and coverage, enrichment profiles, and performance of individual sequences for an engineered transcription factor SHGP based on mazE.

[00157] This figure follows the same format as Fig. 25, illustrating the diversity, enrichment, and performance of an error-prone PCR library for an engineered transcription factor SHGP based on mazE. For detailed explanations of Fig. 30 Parts A, B, and C, please refer to the description of Fig. 25. The sequences shown in Fig. 30 Part C correspond to SEQ ID NOs: 135-154.

[00158] Fig. 31. Exemplary plots illustrating library diversity and coverage, enrichment profiles, and performance of individual sequences for an engineered transcription factor SHGP based on relB.

[00159] This figure follows the same format as Fig. 25, illustrating the diversity, enrichment, and performance of an error-prone PCR library for an engineered transcription factor SHGP based on relB. For detailed explanations of Fig. 31 Parts A, B, and C, please refer to the description of Fig. 25. The sequences shown in Fig. 31 Part C correspond to SEQ ID NOs: 155-174.

[00160] Fig. 32. Exemplary plots illustrating library diversity and coverage, enrichment profiles, and performance of individual sequences for an engineered transcription factor SHGP based on mlB.

[00161] This figure follows the same format as Fig. 25, illustrating the diversity, enrichment, and performance of an error-prone PCR library for an engineered transcription factor SHGP based on rnlB. For detailed explanations of Fig. 32 Parts A, B, and C, please refer to the description of Fig. 25. The sequences shown in Fig. 32 Part C correspond to SEQ ID NOs: 175-194. [00162] Fig. 33. Exemplary plots illustrating library diversity and coverage, enrichment profiles, and performance of individual sequences for an engineered transcription factor SHGP based on yefM.

[00163] This figure follows the same format as Fig. 25, illustrating the diversity, enrichment, and performance of an error-prone PCR library for an engineered transcription factor SHGP based on yefM. For detailed explanations of Fig. 33 Parts A, B, and C, please refer to the description of Fig. 25. The sequences shown in Fig. 33 Part C correspond to SEQ ID NOs: 195-214.

[00164] Fig. 34. Exemplary 2D Heatmap of Motif Enrichment Score for exemplary antibody-like Synthetic Helper Gene Protein sequence library.

[00165] This figure presents a two-dimensional histogram heatmap visualizing the relationship between amino acid motifs of length 5 and their associated log 10 enrichment scores within exemplary antibody-like Synthetic Helper Gene Protein sequences.

[00166] The x-axis represents individual motifs, sorted in descending order based on their calculated motif scores. Each x-position corresponds to a unique 5-amino-acid motif. The y-axis represents the loglO of the enrichment score, ranging from approximately -2 to 2.5.

[00167] The intensity of each point in the heatmap corresponds to the frequency of occurrence, with lighter areas indicating higher frequency and darker areas indicating lower frequency (though the background is white). The color intensity is scaled logarithmically to better visualize the distribution across a wide range of frequencies.

[00168] This visualization method allows for the simultaneous display of motif prevalence and associated enrichment scores within a given SHG category. The solid blocks at left indicate the motifs that cover the canonical, unmutated parent sequence. Vertical structures in the plot indicate motifs that are associated with a wide range of enrichment scores, while horizontal bands suggest enrichment score ranges that are common across multiple motifs.

[00169] This figure provides insights into the distribution of enrichment scores across different motifs in the SHG protein products, allowing for the identification of highly enriched motifs and patterns in the enrichment score distribution.

[00170] Fig. 35. Flow Cytometry Histogram Illustrating Increased Transduction Efficiency

[00171] This figure presents flow cytometry data comparing the transduction efficiency of AAV-SHG-GFP libraries to a control AAV-GFP preparation. The histograms show cell counts (Y-axis, logarithmic scale from 10^A0 to 10^A5) versus relative fluorescence intensity (X- axis). Fig. 35 Parts A and B correspond to Nanobody libraries from SEQ ID NO: 215 and 216, respectively. These libraries underwent 5 rounds of enrichment (as detailed in Figs. 16-19, 22- 25) before being used to transduce fresh reporter cells. A control AAV preparation, created without SHG, was used for comparison. Transduction conditions were set such that the control AAV-GFP preparation was applied at lOx higher titer compared to the AAV-SHG-GFP preparation.

[00172] Three days post-transduction, cells were trypsinized and analyzed by flow cytometry to quantify transduction events by measuring GFP-expressing fluorescent cells. In the histograms, control AAV-GFP results are shown in light gray, while AAV-SHG-GFP results are displayed in dark gray. Both plots demonstrate comparable or superior transduction performance for the AAV-SHG-GFP preparations (more dark gray bars further to the right). Importantly, this equivalent or enhanced performance was achieved with the AAV-SHG-GFP at one-tenth the viral titer compared to the control transduction.

[00173] These results conservatively suggest that the average SHG protein in this library confers at least a 10-fold increase in biological titer compared to the reference AAV preparation. This substantial enhancement in transduction efficiency highlights the unexpected ability of SHGPs for enhancing AAV transduction efficiency and potency (i.e. a lower titer is needed to achieve a comparable biological effect). This addresses a key challenge in developing more potent gene therapy formulations.

[00174]

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

[00175] Numerous specific details are set forth in the following description in order to provide a thorough understanding of the present disclosure. These details are provided for the purpose of example and the claimed subject matter may be practiced according to the claims without some or all of these specific details. It is to be understood that other embodiments can be used, and structural changes can be made without departing from the scope of the claimed subject matter. It should be understood that the various features and functionality described in one or more of the individual embodiments are not limited in their applicability to the particular embodiment with which they are described.

[00176] Definitions

[00177] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the present disclosure belongs. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[00178] The term "affinity purification tag" as used herein refers to a specific type of peptide or protein sequence that is genetically grafted onto a target protein, specifically a Synthetic Helper Gene Product (SHG), for the purpose of removing the SHGP from the mammalian host cell lysate to prevent contamination of viral material by the SHGP.

[00179] The affinity purification tag may be appended to the SHGP at the N-terminus, C-terminus, or may be an internal tag located at various sites within the protein. This tag facilitates the selective separation and removal of the SHGP from other components of the mammalian host cell and the desired viral particles.

[00180] The affinity purification tag operates through specific binding interactions with a corresponding affinity matrix or binding partner, such as immobilized metal ions, antibodies, nanobodies, streptavidin, or other specific molecules, allowing the selective isolation of the tagged SHG. Examples of known affinity tags suitable for this purpose include but are not limited to His-tag, GST, MBP, CBP, Strep-tag, FLAG-tag, HA-tag, SBP-tag, Softag 1, Softag 3, polyarginine tag, polyglutamate tag, and innovative systems like SpyTag, intein-based tags, and immunoaffinity tags like ALFA-tag. Some of these tags may also serve dual roles as solubilization agents or have specific and reversible or cleavable binding properties.

[00181] Typically, the tag is designed to enable purification through affinity techniques that take advantage of these binding characteristics to a corresponding affinity matrix or ligand. This allows the SHG to be efficiently separated and removed from host cell and viral proteins present in cell lysate, while untagged host cell proteins, viral proteins, and desired viral particles may proceed to standard downstream purification without contaminating SHGPs.

This process is integral to removing a potential process-related impurity from the viral production process.

[00182] This approach is particularly useful for large-scale purification during downstream manufacturing processes and is consistent with well-established practices in the field of protein expression and purification within mammalian host cells. It leverages the diverse range of available affinity tags, employing them in a targeted and efficient manner, adapting to specific requirements of the purification process, the nature of the protein, and the desired final purity.

[00183] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide” includes one or more peptides, or mixtures of peptides. Also, and unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

[00184] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X. [00185] The terms “level” or “levels” are used to refer to the presence and/or amount of a target, e.g., a substance or an organism that can be determined qualitatively or quantitatively. A “qualitative” change in the target level refers to the appearance or disappearance of a target that is not detectable or is present in samples obtained from normal controls. A “quantitative” change in the levels of one or more targets refers to a measurable increase or decrease in the target levels when compared to a normal control.

[00186] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[00187] As used herein, the term “mammalian cell” includes host cells from any member of the order Mammalia, such as, for example, human cells, mouse cells, hamster cells, etc. Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK 293, a HeLa cell, or a HT1080 cell. Mammalian cells include mammalian cell cultures which can be either adherent cultures or suspension cultures. Adherent cultures refer to cells that are grown on a solid support surface, for example, on a plastic plate, or other suitable cell culture growth platform. Suspension cultures refer to cells that can be maintained in, for example, culture flasks or other vessels without attachment to a surface, which offers a large surface area for gas and nutrient exchange. Exemplary host cells useful for methods and compositions of the present invention include HEK 293 cells, HEK 293 T cells, Expi293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, HeLa S3 cells, PER.C6 cells, HKB11 cells, CAP cells, Baby Hamster Kidney fibroblasts (BEK cells) (e.g., BEK-21 cells), mouse myeloma cells (e.g., Sp2/0 cells, NSO cells), green African monkey kidney cells (e.g., COS cells and Vero cells), A549 cells, rhesus fetal lung cells (e.g., FRhL-2 cells), or a derivative of any thereof cells. [00188] As used herein, the term “adeno-associated virus (AAV)” refers to a small, replicative-defective, nonenveloped virus which belongs to the genus Dependoparvovirus and the family Parvoviridae. Over 10 adeno-associated virus serotypes have been identified so far, including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In addition to these serotypes, AAV pseudotypes have been developed, where AAV pseudotype contains the capsid of a first serotype and the genome of a second serotype. In addition, there are many AAV vectors derived from the classical serotypes. In addition, there are many animal-derived AAV vectors, including bovine AAV, primate AAV, equine AAV, ovine AAV, canine AAV, mouse AAV, rate AAV, avian AAV, and others. In addition, there are synthetic serotypes that are the result of directed evolution or artificial intelligence design that do not fit into phylogenetic categories or have negligible homology to naturally occurring AAV serotypes. In addition, there are chimeric AAVs that may contain sequence identity of multiple serotypes. An example is an AAV that has AAV2 capsid, but AAV8 ITRs. Another example is an AAV that has a chimeric capsid derived from AAV3, AAV4, and AAV5, while the ITRs are derived from AAV6. The term “AAV vector” as used herein refers to an active, infectious form of the AAV (i.e., viral particle or virion), which is used for delivery the DNA sequence operably linked to one or two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) into infecting cell. Among helper viruses that help AAV to replicate in host cells are adenoviruses, herpesviruses, or papillomaviruses. [00189] The canonical AAV genome is composed of a linear single- stranded DNA molecule which contains approximately 4681 bases. The genome includes inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) at each end which function in cis as origins of DNA replication and as packaging signals for the virus. The ITRs (SEQ ID NO: 8-9) are approximately 145 bp in length. Inverted terminal repeats flank the unique coding nucleotide sequences for the non- structural replication (Rep) proteins (SEQ ID NO: 1-4, 10, 12, 14) and the structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the capsid (SEQ ID NO: 5- 7, 11, 13, 15). The terminal 145 nucleotides are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex as well as genome packaging, integration, rescue. [00190] The term “recombinant” as applied to an AAV or AAV vectors, refers to the virus or vector that is the product of various non-natural, human-made manipulations, such as genetic alterations (such as encapsulation of a heterologous nucleotide sequence of interest), propagation in non-natural environment, and other procedures that result in a virus or vector that is distinct from a virus or vector found in nature. In preferred embodiments, AAV vectors used herein are recombinant AAV vectors, referring to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell (payload). In general, the heterologous polynucleotide is operably linked to two AAV inverted terminal repeat sequences (ITRs) (SEQ ID NO: 8-9). The other components needed for production of recombinant AAV vectors are provided in trans, for example from plasmids, helper viruses, or packaging cell genome.

[00191] As used herein, the term "AAV replication gene" refers to a gene that is involved in the replication and regulation of the adeno-associated virus (AAV) genome, ensuring the efficient replication, packaging, and maintenance of the viral genome. In some embodiments, the AAV replication gene is selected from the group consisting of Rep78, Rep68, Rep52, and Rep40 (SEQ ID NO: 1-4). Rep78 and Rep68 encode large multifunctional proteins with overlapping functions, including DNA binding, helicase activity, and ATPase activity. They are primarily responsible for initiating viral DNA replication and regulating various stages of the AAV life cycle. Rep52 and Rep40 encode smaller isoforms, each enhancing efficiency of DNA replication, playing role in packaging of viral DNA into capsids. By selecting the appropriate AAV replication gene, one can manipulate and control AAV replication dynamics. The choice of AAV replication gene influences factors such as replication efficiency, viral genome copy number, and the ability to establish persistent infection.

[00192] As used herein, the term "AAV capsid encoding gene" refers to a gene that encodes a structural protein (essential component) of the AAV capsid, which is responsible for encapsulating the viral genome and/or facilitating viral entry into target cells. In some embodiments, the AAV capsid encoding gene encodes a protein that is selected from the group consisting of VP1, VP2, and VP3 (SEQ ID NO: 5-7). VP1, VP2, and VP3 are the major capsid proteins of AAV. These proteins self-assemble to form the icosahedral capsid structure, providing stability and protection to the viral genome during transmission and infection. In some embodiments, the AAV capsid encoding gene also encodes an accessory protein, such as the Assembly-Activating Protein (AAP) or MAAP, which play a role in the capsid-assembly process and influence the final capsid structure. By selecting the appropriate AAV capsid encoding gene, one can customize and engineer the AAV capsid, influencing vector tropism, immunogenicity, and other characteristics. The choice of AAV capsid encoding gene impacts the specific properties and behavior of the resulting AAV vector in terms of target cell specificity and transduction efficiency.

[00193] As used herein, the term "AAV helper gene" refers to a gene that is required for the replication, transcription, or packaging of an AAV viral vector in addition to proteins encoded by AAV replication gene and AAV capsid encoding gene. AAV viral helper genes can be classified into two categories: essential helper genes and non-essential helper genes. Essential helper genes are indispensable for replication, transcription, or packaging of the viral vector, while non-essential helper genes enhance the efficiency of vector production without being mandatory for these processes. As used herein, "AAV helper gene" refers to an essential AAV helper gene (SEQ ID NO: 16-23). Adenoviruses are a common source of essential AAV helper genes. Examples of essential adenoviral AAV helper genes include, but are not limited to, Adenovirus El A, Adenovirus E1B55K, Adenovirus E2A, Adenovirus E4orf6, and Adenovirus VA, which play vital roles in facilitating replication, transcription, and packaging of adenoviruses and AAV. AAV helper genes can be derived from various viruses, including but not limited to herpes simplex viruses (Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells; 1979), Human Papillomavirus (Productive Replication of Adeno-Associated Virus Can Occur in Human Papillomavirus Type 16 (HPV- 16) Epi some-Containing Keratinocytes and Is Augmented by the HPV-16 E2 Protein; 2000), Vaccinia virus (Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helper virus dependent parvovirus; 1986), hepatitis B virus (Hepatitis B virus infection enhances susceptibility toward adeno-associated viral vector transduction in vitro and in vivo; 2014), Human Bocavirus (Human Bocavirus 1 Is a Novel Helper for Adeno-associated Virus Replication; 2017), recombinant baculoviruses engineered to express helper genes from the previous viruses (A Recombinant Baculovirus Efficiently Generates Recombinant Adeno-Associated Virus Vectors in Cultured Insect Cells and Larvae; 2018). For, example, HSV-derived AAV helper genes include genes encoding HSV helicase-primase complex (UL5, UL8, UL52) and the major DNA-binding protein (UL29), which have been shown to provide sufficient helper gene function for AAV replication (A subset of herpes simplex virus replication genes provide helper functions for productive adeno-associated virus replication; 1991) (SEQ ID NO: 30-33). In another example, human papillomavirus-derived AAV helper genes include the gene encoding HPV El protein, or HPV El, E2, and E6 genes. In yet another example, Herpes Simplex Virus (HSV)-derived AAV helper genes include genes such as UL5 (Helicase- Primase Complex), UL8 (Helicase-Primase Complex), ISHGP8 (Single-strand DNA-binding protein), and ISHGP27 (Transcriptional regulator). In yet another example, Herpes Simplex Virus (HSV)-derived AAV helper genes include genes such as p80 (Late expression factor), pl43 (DNA replication factor), p40 (Nucleocapsid assembly factor), and p32 (Single-strand DNA-binding protein). AAV helper genes derived from other viruses, or potentially obtained through artificial intelligence, can also be utilized in the methods disclosed herein. [00194] As used herein, the term "Synthetic Helper Gene" (SHG) refers to a synthetic (i.e., non-naturally occurring) gene construct that enhances viral vector production when expressed in host cells (i.e. cells configured to produce viral particles), but that is not essential to viral vector production. In some preferred embodiments, SHGs are genes that encode antibody-like proteins, including but not limited to engineered antibody mimetics (e.g. Affibodies, DARPins, Monobodies, etc...) and engineered single-domain antibodies (e.g. nanobodies and single-chain variable fragments (scFvs), etc. . .). In other preferred embodiments, SHGs are genes that encode engineered transcription factor proteins, such as transcriptional activators or repressors. When expressed in viral-producing host cells, Synthetic Helper Genes produce a Synthetic Helper Gene Product (SHGP), which is a protein that modulates the virus-producing host cell and increases viral titer and/or transduction efficiency of the resulting viral composition by at least 2-fold compared to a reference viral composition produced by host cells without the Synthetic Helper Gene, under essentially identical packaging and transduction conditions (such as when no modifications are made to the viral capsid, and no additives are included into the transduction).

[00195] The term "Synthetic Helper Gene Product" (SHGP) refers to a non-naturally occurring polypeptide produced by expression from a Synthetic Helper Gene nucleotide sequence and present in virus-producing cells. SHGPs are limited to engineered binding proteins as described above and do not include nucleotide products (e.g., sgRNAs, siRNAs). SHGPs are ribosomally expressed polypeptides that do not occur in nature (i.e., differ by at least one amino acid), are not essential for viral production (i.e., they are not essential viral replicative genes), typically range from 30 to 300 amino acids in length, are preferably expressed intracellularly during viral production (though may be added exogenously), are neither incorporated into nor essentially present in the final viral preparation. SHGPs function by manipulating cellular behavior of virus-producing host cells during viral packaging through binding to various molecular targets, which modulates cellular processes and drives differential cellular phenotypes that result in enhanced viral vector production. [00196] SHGPs comprise two classes of binding proteins. The first class is antibody -like proteins, which include, but are not limited to, engineered antibody mimetics such as Affibodies, DARPins, Monobodies, Anticalins, Affimers, Alphabodies, and Centyrins, as well as engineered single-domain antibodies including nanobodies and scFvs. These proteins typically contain between 5 and 40 specificity-determining residues located on surface-exposed regions and arranged to form a binding pocket, interface, or grouped into one or more complimentary determining regions (CDRs). The second class of SHGPs are engineered transcription factor proteins. Such proteins typically contain one or more DNA-binding motifs such as a Helix-Turn-Helix (HTH), Zinc Finger, Leucine Zipper (bZIP), Helix-Loop-Helix (HLH), or Homeodomain. These proteins must include a nuclear localization signal (NLS), if not already present, to ensure efficient translocation to the nucleus of the packaging cell.

[00197] SHGPs explicitly exclude any naturally occurring amino acid sequence, any non-amino acid polymer (e.g., sgRNAs, siRNAs), any full-length antibody sequence (e.g., all heavy /light chains), any sequence over 300 amino acids, proteins or their coding sequences that are essentially packaged into or incorporated into the viral particle for enhanced viral titer and/or transduction, proteins or their coding sequences that must be present in the viral composition for increased transduction performance (e.g., transduction enhancers), any protein essential for viral production (e.g., standard viral packaging proteins that are known to those in the art), and any protein who’s ability to improve viral production requires addition of a second effector compound (i.e. it is not an inducible expression system). In preferred embodiments, the critical attributes of Synthetic Helper Gene Products are their expression in viral packaging cells, their ability to effectively manipulate biological systems for optimized viral production and/or their ability to enhance the infectiousness of the produced viral vector compositions.

[00198] In preferred embodiments of the disclosed methods and compositions, Synthetic Helper Gene Products are ribosomally expressed polypeptides.

[00199] In preferred embodiments of the disclosed methods and compositions, a Synthetic Helper Gene Product increases manufacturability of a viral vector composition produced in the presence of the Synthetic Helper Gene Product, such as increases viral titer and/or transduction efficiency of the viral vector composition by at least two-fold in comparison to a reference viral vector composition produced under essentially identical conditions but in the absence of the Synthetic Helper Gene Product. In preferred embodiments, once a viral vector composition is produced in the presence of the Synthetic Helper Gene Product in host cells, the viral vector composition may be purified from the host cells and used to infect target cells in the absence of the Synthetic Helper Gene Product. In other words, Synthetic Helper Gene Product is not essentially present in a viral vector composition of increased viral titer and/or transduction efficiency once the viral vector composition is produced in the presence of the Synthetic Helper Gene Product in host cells and purified from the host cells. The term “not essentially present” refers to embodiments, where viral vector composition does not contain any Synthetic Helper Gene Product molecules, or contain only trace amounts of Synthetic Helper Gene Product (SHGP) molecules, such as when viral vector composition is purified from host cells containing SHGP molecules after production, but a small amount of SHGP molecules (e.g., less than 1% of SHGP molecules) is still retained within the viral vector composition. In preferred embodiments, Synthetic Helper Gene Product is required during manufacturing of the viral vector composition of increased viral titer and/or transduction efficiency which may comprise a payload but is not required during further use of the produced viral vector composition of increased viral titer and/or transduction efficiency, such as during delivery of the payload to target cells. In preferred embodiments, Synthetic Helper Gene Product is not present, or not essentially present, in the viral vector composition during transduction to target cells.

[00200] In preferred embodiments, Synthetic Helper Gene Product is not attached to a viral capsid (is not attached to any viral protein) of the virus or viral vector composition during or after obtaining of the viral vector composition. In preferred embodiments, SHGP is structurally different from any one of viral replication genes, viral structural genes or additional viral genes necessary to produce the virus in the host cells (SHGP is not essential for viral production). In preferred embodiments, viral genes that encode proteins of the viral capsids of the virus used for production of the viral vector composition are not altered, mutated or modified, such as there are no structural modifications in viral proteins of the viral capsids of the claimed viral vector composition. In preferred embodiments, SHGP or SHG is not cytotoxic, cytostatic and does not otherwise interfere with cell growth of the host cells that contain viral vectors produced in the present of SHGP. In preferred embodiments, SHGP is a ribosomally expressed polypeptide, present only during viral packaging, which results in an increase in viral yield/quality of the viral vector composition produced in the presence of SHGP, but SHGP is not present, or essentially present, in the resulting formulation of the viral vector composition.

[00201] As used herein, the term "lentiviral gag gene" (SEQ ID NO: 26) refers to a gene that participates in a lentivirus assembly in host cells and typically encodes a structural protein. As used herein, the term "lentiviral pol gene" (SEQ ID NO: 25) refers to a gene that encodes an enzyme required for reverse transcription and/or integration of lentivirus into the host cell genome. As used herein, the term "lentiviral rev gene" (SEQ ID NO: 24) refers to a gene that facilitates nuclear export of unspliced or partially spliced viral RNAs in host cells. As used herein, the term "env gene" (SEQ ID NO: 27) refers to an envelope gene that participates in a lentivirus assembly in host cells and encoding a glycoprotein from an enveloped virus.

[00202] The term “synthetic” as used in reference to a nucleic acid molecule or to a polypeptide molecule, e.g., a Synthetic Helper Gene Product, refers to molecules that are created by human intervention and/or they are non-naturally occurring. A synthetic nucleic acid sequence can include any type of modification that can be made to a nucleic acid (e.g., introduction, substitution, deletion, replacement, rearrangement, epigenetic modification, etc.). In some embodiments, a Synthetic Helper Gene Product may be selected or determined by the methods disclosed herein and then may be further modified to obtain a further Synthetic Helper Gene Product. In some embodiments, a further Synthetic Helper Gene Product has one or more improved characteristics compared to the starting Synthetic Helper Gene Product, for example, increased membrane permeability or increased stability in host cells. Sequence of a further Synthetic Helper Gene Product can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid differences (e.g., substitutions and/or additions) compared to the sequence of starting Synthetic Helper Gene Product. A further Synthetic Helper Gene Product generally exhibits at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding starting Synthetic Helper Gene

Product. Non-naturally occurring amino acids as well as naturally occurring amino acids are included within the scope of permissible substitutions or additions. The term "synthetic" in the context of Synthetic Helper Gene Product is not to be construed as imposing any condition for any particular starting composition or method by which the Synthetic Helper Gene Product is created. Thus, Synthetic Helper Gene Product denotes a composition and not necessarily a product produced by any given process.

[00203] In some embodiments, variants of a Synthetic Helper Gene Product (such as a further Synthetic Helper Gene Product described above) displaying only non-substantial or negligible differences in structure and/or sequence can be generated by making conservative amino acid substitutions in the Synthetic Helper Gene Product. By doing this. Synthetic Helper Gene Product variants that comprise a sequence having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with the Synthetic Helper Gene Product sequences can be generated, retaining at least one functional activity of the Synthetic Helper Gene Product, e.g., ability to increase viral titer and/or transduction efficiency of a viral vector composition. Examples of conservative amino acid changes are known in the art. Examples of non-conservative amino acid changes that are likely to cause major changes in peptide structure are those that cause substitution of (a) a hydrophilic residue, e.g., serine or threonine, for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, for (or by) an electronegative residue, e.g., glutamic acid or aspartic acid; or (d) a residue having a bulky side chain, e.g., phenylalanine, for (or by) one not having a side chain, e g., glycine. Methods of making targeted amino acid substitutions, deletions, truncations, and insertions in peptides are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for polynucleotide alterations are well known in the art, for example, Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192 and the references cited therein.

[00204] The term “sequence identity” is a measure of identity between peptides at the amino acid level, and a measure of identity between nucleic acids at nucleotide level. The peptide sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned. "Sequence identity" means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. For example, the BLAST algorithm (NCBI) calculates percent sequence identity and performs a statistical analysis of the similarity and identity between the two sequences. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. Another program that can be used to calculate sequence identity is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.

[00205] The terms “corresponding to position(s)” or “position(s) . . . with reference to position(s)” of or within a peptide or a polynucleotide, such as recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions of a disclosed sequence, such sequence set forth in the Sequence Listing, refers to nucleotides or amino acid positions identified in the polynucleotide or in the peptide upon alignment with the disclosed sequence using a standard alignment algorithm, such as the BLAST algorithm (NCBI). One skilled in the art can identify any given amino acid residue in a given peptide at a position corresponding to a particular position of a reference sequence, such as set forth in the Sequence Listing, by performing alignment of the peptide sequence with the reference sequence (for example, by using BLASTP publicly available through the NCBI website), matching the corresponding position of the reference sequence with the position in peptide sequence and thus identifying the amino acid residue within the peptide.

[00206] The term “host cell” refers to a mammalian or insect cell. The term “host cell permissive for AAV replication" refers to a cell, such as a mammalian or insect cell, in which AAV can replicate and generate AAV vectors when certain elements necessary for intracellular AAV replication are present or introduced into such a cell. Elements necessary for intracellular AAV replication, packaging and/or vector generation include AAV replication gene(s), AAV capsid encoding gene(s), and viral helper gene(s). Since AAV is replication-defective, specific viral helper gene(s) that originated from adenoviruses, herpesviruses (HSV), bocaviruses or papillomaviruses need(s) to be inserted into a host cell to make it permissive for AAV replication. In exemplary embodiments, viral helper gene is an adenovirus helper gene. In other embodiments, viral helper gene is HSV helper gene, bocavirus helper gene, or a papillomavirus helper gene. As referred to herein, the term “adenovirus helper gene” refers to a gene that is composed of one or more nucleic acid sequences derived from one or more adenovirus subtypes or serotypes that contributes to AAV replication, packaging and/or generation of AAV vectors.

[00207] In some embodiments, AAV vectors produced in host cells by the methods disclosed herein are used as therapies themselves. In some embodiments, produced AAV vectors are used in the research, production, and/or manufacturing processes that can generate therapies. For example, AAV vectors can be used in many ways that include but are not limited to vaccines, cancer therapies (e.g., oncolytic therapies), and/or gene therapies (e.g., in vivo gene and/or genomic editing). Methods of the present disclosure can be used to generate host cells with beneficial characteristics for expression of an AAV vector. Methods of the present disclosure can be used to generate helper viruses with beneficial characteristics for production of an AAV vector. Methods of the present disclosure can be used to generate packaging plasmid sets with improved/beneficial characteristics for production of an AAV vector.

[00208] In preferred embodiments, a host cell provided herein includes one or more one or more AAV replication genes encoding non-structural replication (Rep) (SEQ ID NO: 1-4, 10, 12, 14) proteins (such as, for example and without limitation, Rep 78 (SEQ ID NO: 4), Rep 68 (SEQ ID NO: 3), Rep 52 (SEQ ID NO: 2) and Rep 40 (SEQ ID NO: 1)); one or more AAV capsid encoding genes that encode structural (VP) proteins (such as, without limitation, VP1, - 2 and -3) forming the AAV capsid; and one or more viral helper genes (such as, without limitation, Adenovirus El A, Adenovirus E1B55K, Adenovirus E2A, Adenovirus E4orf6, and Adenovirus VA). Viral helper genes may include various adenoviral virus genes, HSV genes, bocavirus genes and papillomavirus genes. These genes (e.g., AAV replication genes, AAV capsid encoding genes, and/or viral helper genes) are inserted into a host cell operable linked to (under control of) other transcriptional regulatory sequences, including promoters (e.g., regulatable promoters). Exemplary description of regulatory sequences including suitable promoters for use in the disclosed methods can be found in US 20200199627 Al and US 6924128 B2, incorporated herein. In some embodiments, two or more AAV replication genes, AAV capsid encoding genes, and/or viral helper genes may be utilized simultaneously in the disclosed methods to produce AAV vectors.

[00209] In some embodiments, one or more nucleic acid sequences essential for production of AAV vectors in host cells comprise a heterologous promoter sequence that is or comprises an SV40 promoter, an elongation factor (EF)-l promotor, a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK)l promoter, a ubiquitin (Ubc) promoter, a human beta actin promoter, a tetracycline response element (TRE) promoter, a spleen focus-forming virus (SFFV) promoter, a murine stem cell virus (MSCV) promoter, a supercore promoter (SSHGP), a CAG promoter, or a derivative thereof. In some embodiments, one or more nucleic acid sequences essential for production of AAV vectors in host cells comprise a heterologous enhancer sequence that is or comprises a CMV early enhancer, a cAMP response-element (CRE) enhancer, or a derivative thereof. In some embodiments, one or more nucleic acid sequences essential for production of AAV vectors in host cells can be integrated into a mammalian cell genome and under the control of an inducible transcriptional control element (e.g., inducible promoter and/or inducible enhancer). In some embodiments, one or more nucleic acid sequences essential for production of AAV vectors in host cells can be present episomally in a mammalian cell and under the control of an inducible transcriptional control element (e.g., inducible promoter and/or inducible enhancer).

[00210] In some embodiments, the elements necessary for intracellular AAV replication, packaging and/or vector generation in a host cell are contained within the host cell in separate nucleic acid molecules, for example separate chromosomes, plasmids, or vectors. In other embodiments, the nucleic acid molecules encoding the various elements necessary for AAV replication, packaging and/or vector generation are included on the same chromosome, plasmid, or vector. In further embodiments, certain of the elements are contained on the same nucleic acid molecule (e.g., AAV capsid encoding genes and AAV replication genes), while other genes are contained on separate nucleic acid molecules (e.g., helper genes). In yet other embodiments, certain of the elements are integrated into genome of the host cell.

[00211] The term “manufacturability" refers to the degree to which a product (e.g., AAV vector for gene therapy) can be effectively manufactured given its design, cost, purity, yield, safety, and efficacy requirements. Manufacturability is centered on a) overall feasibility, e.g. rAAV that works when produced at lab scale fails to work when produced at larger scale for any one or combination of reasons including, but not limited to, higher toxicity, lower safety, lower viral titer, lower potency/transduction efficiency, tropism, higher contamination, higher impurities (product and process-related), immunogenicity, higher purification requirements, stability, downstream processing requirements, batch failure rate,; and b) excess cost, e.g. the intrinsic inefficiency of rAAV production can result in products that cost more to manufacture than they can be sold for.

[00212] In the context of AAV manufacturing, the most common manufacturability challenges include: viral titer (as measured by VG/ml or VG/cell); fulkempty capsid ratio (commonly assessed by comparing the genome copy number, or physical titer, to the total viral particle counts based on capsid protein); infectious unit titer (as measured by lU/ml or lU/cell), also referred to as transducing unit titer (as measured by TU/ml or TU/cell); a related feature is transduction efficiency, or the TU:VG ratio, which indicates how many functional AAV vectors are contained out of the total number of full, genome-containing AAV vectors. The term “Infectious unit titer” as used herein is a measurement of the number of viral particles that can transduce cells (e.g. per cell or per ml; provided as lU/ml or lU/cell). Infectious unit titers are typically quantified with cell transduction assays (e.g. FACS or fluorometric microscopy on transduced cells, TCID50).

[00213] The terms “reference host cell”, “reference plurality of host cells” as used herein refer to a host cell or a plurality of host cells, respectively, not comprising a Synthetic Helper Gene Product, according to various embodiments of the present invention. Similarly, “reference AAV vector composition” as used herein refers to an AAV vector composition produced in host cell in the absence of a Synthetic Helper Gene Product (reference AAV vector composition have the same serotype or pseudotype as the AAV vector composition to which reference AAV vector composition is compared). These terms are used to designate standard or control host cells (or AAV vector composition), which are not modified by a Synthetic Helper Gene Product. Reference AAV vector composition is produced from host cells under identical, or nearly identical conditions, as the AAV vector composition to which reference AAV vector composition is compared, except from absence of Synthetic Helper Gene Products in the host cells during production (reference AAV vector composition is produced from the same host cells, but without Synthetic Helper Gene Products). In some embodiments, reference or control host cells (or AAV vector composition) are tested substantially simultaneously with the testing host cells of interest (e.g., host cells comprising a Synthetic Helper Gene Product). Typically, as would be understood by those skilled in the art, reference or control host cells (or AAV vector composition) are characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[00214] As used herein, the term “infectivity” refers to the ability of the viral vector to enter and successfully transduce target cells. Optimizing viral production in cells, such as by expressing a Synthetic Helper Gene Product (SHGP), may improve infectivity by generating viral particles with enhanced capsid structures that facilitate cellular entry and successful delivery of the genetic payload. An optimized cell environment for viral production may influence various intracellular processes that contribute to the overall quality of the viral particles. These processes may include post-translational modifications like glycosylation or sumoylation, methylation of viral payload DNA, the activity of various proteases in capsid maturation, and vesicular pH, which has been shown to play a crucial role in capsid processing. By optimizing these cell behaviors and pathways, the quality of the viral particles can be improved, resulting in enhanced infectivity and better clinical outcomes.

[00215] As used herein, the term “transduction efficiency” refers to the effectiveness (efficiency) of viral particles in delivering their genetic payload to target cells. This efficiency may be represented by the transducing units to vector genomes ratio (TU:VG). Viral formulations with enhanced transduction efficiency can achieve greater numbers of DNA delivery events for the same number of DNA-containing viral particles. For example, if formulation A requires 10 viral particles to achieve one transduction event (transduction efficiency of 0.1) and formulation B requires 5 viral particles to achieve on transduction event (transduction efficiency of 0.2), then formulation B would have a 2 fold greater transduction efficiency compared to formulation A (0.2 / 0.1 = 2). Enhanced transduction efficiency may be achieved by optimizing the cellular environment for viral production, leading to viral particles with improved capsid structure, viral particle biochemistry, or viral particle assembly.

Increasing transduction efficiency refers to improvements in the TU:VG ratio, which encompasses both infectivity and biological titer. Higher transduction efficiency implies a higher proportion of infectious particles to total vector genomes, which can result from a higher biological titer and/or lower VG levels.

[00216] As used herein, the term “viral titer” refers to the concentration or quantity of viral particles present in a given sample. By measuring the viral titer of a given sample, one can gain valuable insight into the manufacturability and clinical utility of viral compositions. Viral titer measurements typically fall into two broad categories: physical titer or biological titer (each defined separately). Viral titer is reported using different metrics depending on what type of virus is being evaluated and what aspect of the viral material is being measured. In the context of AAV, physical titer is often reported in viral genomes per milliliter (vg/ml) or genome copies per milliliter (gc/ml) and can be measured using techniques like qPCR or ddPCR; however, biological titer is becoming an increasingly important metric as the clinical use of AAV matures. In the context of lentivirus, biological titers are more commonly reported with transducing units per milliliter (TU/ml) or infectious units per milliliter (lU/ml) being typically used metrics; however, lentiviral physical titers are also commonly reported in viral particles per milliliter (vp/ml). This titer is often determined by measuring the amount of p24 antigen, a viral protein, in the sample via ELISA or by quantifying the amount of viral RNA present using qRT-PCR.

[00217] As used herein, the term “physical titer” refers to the total count of viral particles in a sample, irrespective of their infectivity. This is generally quantified by assessing a component of the viral particle, such as viral RNA, DNA, or protein(s). Physical titer may be reported in a variety of ways depending both on the conventions for a given virus and the methods by which physical titer is determined. The most commonly reported physical titer metrics are viral genomes per milliliter (vg/ml), genome copies per milliliter (gc/ml), or viral particles per milliliter (vp/ml). In the context of AAV, qPCR or ddPCR are commonly used to determine and report vg/ml or gc/ml. In the context of lentivirus, vp/ml is commonly reported and is often determined by measuring p24 antigen by ELISA or quantifying viral RNA by qRT-PCR. For herpes simplex virus (HSV), the physical titer is typically reported in gc/ml; adenovirus in vp/ml; and baculovirus in occlusion bodies per milliliter (OBs/ml) or vp/ml for occluded and non-occluded baculoviruses respectively.

[00218] As used herein, the term "biological titer" refers to the count of biologically functional viral particles in a sample (i.e., viral particles that are capable of infecting target cells or transducing genes (i.e., payload) into target cells, which may or may not lead to gene expression). Optimizing viral production in cells, such as by expressing a Synthetic Helper Gene Product (SHGP) as disclosed herein, may enhance the biological titer by increasing the proportion of functional viral particles. Higher biological titers may result from optimized cellular environments that support the generation of viral particles with improved capsid structures, tropism, and immune evasion properties. Depending on the virus being measured and specific method used to quantify the viral material, biological titer is typically reported in infectious units per milliliter (lU/ml or IFU/ml), transducing units per milliliter (TU/ml), or plaque-forming units per milliliter (PFU/ml).

[00219] TU/ml is determined by quantifying the number of target cells that express the transgene after being exposed to a known volume of the viral vector preparation. This measurement provides a more functional assessment of the viral vector, as it considers the vector's ability to deliver and express the transgene, e.g., green fluorescent protein (GFP), in target cells. lU/ml represents the ability of the viral vector to infect target cells, regardless of whether it leads to transgene expression or not. It is determined by quantifying the number of target cells that are infected (i.e., contain viral genomes) after being exposed to a known volume of the viral vector preparation. PFU/ml is measured by the ability of a virus to form plaques on a cell monolayer. By counting the number of plaques formed by a given dilution of viral material, the number of PFU/ml can be determined.

[00220] Different viruses and their applications dictate a specific metric to be used for biological titers. In the context of gene therapy, where a virus acts as a vector, the titer may be reported in TU/ml or lU/ml to reflect the number of cells successfully transduced. For example, adeno-associated virus (AAV) biological titers are often reported TU/ml, determined by assays that measure either the ability of the AAV to transduce cells and express a particular gene like green fluorescent protein (GFP). Lentiviral biological titers are also commonly reported as TU/ml or lU/ml, again reflecting either the transducing capability or the general infectivity of the virus. Herpes simplex virus (HSV) or adenovirus, biological titers are often reported as lU/ml, TU/ml, or PFU/ml, based on whether the assay measures general infectivity, gene transduction, or plaque formation.

[00221] As used herein, the term “payload” refers to any entity of interest for delivery by an AAV vector produced by methods of the present disclosure. For example, such a payload may be desired to be introduced into a cell, organ, organism, and/or cells. In some embodiments, a payload sequence is or comprises a heterologous nucleic acid sequence for delivery by an AAV vector. In some embodiments, a payload sequence comprises an encoding region and one or more of a gene regulatory element and a transcription terminator. Nonlimiting examples of gene regulatory elements include promoters, transcriptional activators, enhancers, and polyadenylation signals. In some embodiments, a payload sequence comprises an encoding region, a gene regulatory element, and a transcription terminator, positioned relative to each other such that the encoding region is between the gene regulatory element and the transcription terminator. In some embodiments, a coding sequence encodes a gene product. In some embodiments, the gene product is an RNA molecule. In some embodiments, an encoding region encodes a polypeptide. In some embodiments, the payload may incorporate multiple functional units (e.g., a promoter region, an intron, a Kozak sequence, an enhancer, a polyadenylation sequence, and/or a cleavage sites or sequence that encode a protein). Some payloads may be nucleic acid-based and not encode a protein, such as miRNA, siRNA, or aptamers. For example, AAV vectors may contain as a payload the viral genome, either in whole or in part (e.g., only essential components), of any naturally occurring and/or recombinant AAV serotype nucleotide sequence or variant. The payload may be singlestranded (and containing 2 ITRs (SEQ ID NO: 8-9)) or self-complementary (and containing 3 ITRs (SEQ ID NO: 8-9)), and can be produced or modified using various methods known in the art.

[00222] As used herein, the term “polypeptide” is used interchangeably with “peptide” and refers to a molecule comprising a chain of six or more amino acid residues joined by peptide bonds. In some embodiments, a peptide comprises 6 to 10000 amino acid residues. The term “Synthetic Helper Gene Product” or “SHGP” indicates that the produced synthetic peptide functionally impacts viral production. In some embodiments, a Synthetic Helper Gene Product comprising only natural amino acid residues may be selected by using methods described herein; then, a modified version of the Synthetic Helper Gene Product (comprising one or more modified or non-standard amino acid residues) may be prepared and used to enhance viral production. Various modifications are known in the art to enhance cellular permeability, stability or other properties of the selected SHGPs.

[00223] As used herein, the term "amino acid" refers to an organic compound comprising an amine group, a carboxylic acid group, and a side-chain specific to each amino acid, which serve as a monomeric subunit of a peptide. An amino acid includes the 20 standard, naturally occurring or canonical amino acids as well as non-standard amino acids. The standard, naturally-occurring amino acids include Alanine (A or Ala), Cysteine (C or Cys), Aspartic Acid (D or Asp), Glutamic Acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Tip), and Tyrosine (Y or Tyr). An amino acid may be an L-amino acid or a D-amino acid. Non-standard amino acids may be modified amino acids, amino acid analogs, amino acid mimetics, nonstandard proteinogenic amino acids, or non-proteinogenic amino acids that occur naturally or are chemically synthesized. Examples of non-standard amino acids include, but are not limited to, selenocysteine, pyrrolysine, and N-formylmethionine, P- amino acids, gamma amino acids, delta amino acids, Homo-amino acids, Proline and Pyruvic acid derivatives, 3- substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, N-methyl amino acids.

[00224] As used herein, each amino acid residue used in the disclosed SHGPs may be categorized into five categories based on the properties of their side chains/R-groups: HYD, ARO, POL, POS and NEG. As used herein, 20 standard amino acid residues are categorized as follows:

[00225] 1) HYD group: amino acid residues with hydrophobic/aliphatic/non-polar/non- aromatic R-groups, which consists of residues selected from the following group: 'G', 'A', 'V, T, 'L', 'M', P';

[00226] 2) ARO group: amino acid residues with hydrophobic aromatics R-groups, which consists of residues selected from the following group: 'F, 'W, 'Y';

[00227] 3) POL group: amino acid residues with polar uncharged R-groups, which consists of residues selected from the following group: 'S', 'T', 'Q', 'N', 'C;

[00228] 4) POS group: amino acid residues with positively charged (Basic) R-groups, which consists of residues selected from the following group: 'K', 'R', H'; and

[00229] 5) NEG group: amino acid residues with negatively charged (Acidic) R-groups, which consists of residues selected from the following group: 'D', 'E.

[00230] In some embodiments where non-natural and/or modified amino acid residues are used in a SHGP, such residues may also be categorized into one or more of the same five categories based on the chemical properties of their side chains/R-groups. There are cases where non-natural and/or modified amino acid residues have chemical properties (between two or more categories); in such cases, such residues can be assigned to more than one category (e.g., assigned to both HYD and POL, or to both ARO and POL), see some specific examples below.

[00231] As used herein, the term "post-translational modification" refers to modifications that occur on a peptide after its translation by ribosomes is complete. A post- translational modification may be a covalent modification or enzymatic modification. [00232] As used herein, the term “linker” refers to one or more of a nucleotide, a nucleotide analog, an amino acid, a peptide, a peptide, a polymer, or a non-nucleotide chemical moiety that is used to join two molecules. A linker may be used to join a binding agent with a coding tag, a recording tag with a peptide, a peptide with a support, a recording tag with a solid support, etc. In certain embodiments, a linker joins two molecules via enzymatic reaction or chemistry reaction (e.g., click chemistry). In preferred embodiments, linker amino acids include Gly, Ala, Ser, Thr, and Pro, but may include others, depending on the nature of the domains being joined.

[00233] The term “ligand” as used herein refers to any molecule or moiety connected to the compounds described herein. “Ligand” may refer to one or more ligands attached to a compound. In some embodiments, the ligand is a pendant group or binding site (e.g., the site to which the binding agent binds). For example, a Synthetic Helper Gene may be a ligand to a cellular protein.

[00234] As used herein, the term “barcode” refers to a nucleic acid molecule, such as DNA molecule, of about 3 to about 100 bases that provides a unique identifier tag (identifying information) for a Synthetic Helper Gene Product produced in a host cell. A barcode can be an artificial sequence or a naturally occurring sequence. In certain embodiments, each barcode within a population of barcodes is different. In other embodiments, a portion of barcodes in a population of barcodes is different, e.g., at least about 10%, 50%, 90% of the barcodes in a population of barcodes is different.

[00235] The term “provirus” as used herein refers to the genetic material of a virus that has been integrated into the genome of a host cell. In this integrated state, the virus is not actively replicating and does not produce virions. Instead, the viral genome is passively replicated along with the host genome as part of the normal host replication cycle. For example, AAVs can enter a provirus state where the AAV genome is integrated into the host cell genome. Many viruses are known to have provirus stages of their replication cycle. Proviruses can also be created via genetic engineering, for example via transposon integrations. This is useful for creating proviruses in situations where a virus may not naturally integrate into the host genome.

[00236] In certain conditions, proviruses can become mobilized and rescued to produce viral particles. In some cases, the provirus remains dormant or latent within the host cell. It can be activated by various stimuli such as environmental stress, exposure to certain chemicals, ultraviolet radiation, changes in the host's health, viral infection (e.g. of helper viruses). Activation initiates the transcription of the proviral DNA. [00237] Sometimes, proviruses may be defective and unable to produce new virus particles on their own. They can be "rescued" if the host cell is infected with a similar virus. In this case, the proteins produced by the new infection can package the genetic material of the provirus, leading to the production of viral particles with the proviral genome.

[00238] In the context of AAV, in the absence of a helper virus (like adenovirus or herpesvirus), the AAV can remain latent and not produce any viral particles. However, when the cell is subsequently infected with a helper virus, the AAV provirus can be activated or "rescued.” The helper virus provides necessary factors that initiate the replication of AAV. The AAV genome is then transcribed and translated to produce viral proteins.

[00239] The term “helper virus” as used herein refers to a virus that provides one or more helper functions encoded by one or more helper genes encoded on the helper virus genome. A helper virus allows an otherwise deficient coinfecting virus to replicate. Helper viruses are also commonly used to replicate and spread viral vectors for gene therapy. A helper provides essential functions or factors enabling a replication defective or replication dependent virus to complete its replication cycle within a host cell. The helper virus contributes elements that the defective virus lacks, which are necessary for the synthesis, assembly, and sometimes the release of new viral particles.

[00240] In the case of Adeno-Associated Virus (AAV), it is known as a dependoparvovirus because it requires the help of a helper virus to replicate efficiently. AAV, in its natural state, relies on the presence of helper viruses such as Adenoviruses or Herpes Simplex Viruses. When cells are coinfected with AAV and one of these helper viruses, the helper virus provides essential replication factors that AAV lacks, allowing AAV to undergo a productive infection. Helper viruses can be natural or recombinant, with the exemplary recombinant helper virus being baculovirus expressing Adenoviral helper genes necessary for the production of AAV.

[00241] The term “chimera” or “chimeric virus” as used herein refers to a virus comprising genetic material originating from two or more distinct viruses. For example, lentivirus chimeras are commonly made by substituting the wildtype HIV-1 derived envelope glycoprotein gene with a variety of other glycoprotein gene derived from enveloped viruses. [00242] The term “transfer vector” or “transfer plasmid” as used herein refers to a plasmid that contains a DNA payload sequence intended to be packaged in a viral vector and a packaging sequence. For example, an AAV transfer vector will encode a desired DNA payload flanked by two ITR sequences (SEQ ID NO: 8-9). An exemplary lentiviral transfer vector will contain a desired nucleotide sequence payload operably linked to a Psi packaging sequence (SEQ ID NO: 34). Transfer vectors, at a minimum, require a packaging nucleotide sequence (e.g. AAV ITRs (SEQ ID NO: 8-9), lentiviral Psi sequences (SEQ ID NO:34)) to ensure the nucleic acid is packaged into viral. Depending on the desired behavior of the viral vector, transfer vectors may comprise additional components, including, but not limited to promoters, terminators, regulatory elements, replication genes, capsid encoding genes, helper genes, integration elements, replication sequences (e.g. ITRs (SEQ ID NO: 8-9), LTRs (SEQ ID NO:28-29).

[00243] The term “operably linked” refers to a functional relationship between two or more genetic elements within a nucleic acid molecule. When two genetic elements are operably linked, it means they are connected in such a way that they can interact to perform their intended biological function effectively. For example, when a first nucleotide sequence is operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the first nucleotide sequence within viral capsids, it means that the first nucleotide sequence is placed in the viral genome in such a way to allow the first nucleotide sequence to be efficiently encapsulated within the viral capsids. Specific details of such a placement for a particular virus are known to those skilled in the field of viral production from host cells, such as AAV production, lentiviral production of production of other viruses disclosed herein. Exemplary methods of virus production are disclosed, for example, in US Pat. No. 5,278,056, US Pat. No. 6207455 Bl, US Pat. No. 9057056 B2, US 20040161848 Al, US 20220228129 Al, US 20230111672 Al, US 20210047657 Al, US Pat. No. 11898170 B2, US Pat. No. 7749491 B2, and US Pat. No. 9102943 B2, each incorporated herein by reference.

[00244] The term “nested virus” or “nested viral vector” as used herein refers to a viral composition containing at minimum the transfer vector of a “guest virus” that is linked to a “host virus” as the payload of the host virus and in which the host virus and guest virus genomes are packaged into the host virus viral particle. For example, the transfer vector of guest virus A, may be inserted as a payload into the transfer vector of host virus B and the nested viral genome may be packaged into viral particles of host virus B.

[00245] Disclosure of exemplary methods and compositions.

[00246] In some embodiments, the present teachings disclose methods for selecting genetically encoded, endogenously (ribosomally) expressed Synthetic Helper Gene Products (SHGPs) that enhance AAV vector (or any other virus vector) manufacturability (such as packaging and infectivity of AAV capsids) in comparison with AAV vector (or any other virus vector) manufacturability in the same host cells under essentially the same conditions but in the absence of the SHGP production in these host cells. In preferred embodiments, once a viral vector composition is produced in the presence of the Synthetic Helper Gene Product in host cells, the viral vector composition may be purified from the host cells and used to infect target cells in the absence of the Synthetic Helper Gene Product. In other words, Synthetic Helper Gene Product is not essentially present in a viral vector composition of increased viral titer and/or transduction efficiency once the viral vector composition is produced in the presence of the Synthetic Helper Gene Product in host cells and purified from the host cells. In preferred embodiments, Synthetic Helper Gene Product is required during manufacturing of the viral vector composition of increased viral titer and/or transduction efficiency which may comprise a payload but is not required during further use of the produced viral vector composition of increased viral titer and/or transduction efficiency, such as during delivery of the payload to target cells. In preferred embodiments, viral genes that encode proteins of the viral capsids of the virus used for production of the viral vector composition are not altered or modified, such as there are no modifications in viral proteins of the viral capsids.

[00247] AAV capsid quality is a complex matter. A single AAV viral preparation in host cells contains a heterogeneous mix of viral particles with diverse post translational modification profiles, capsid protein stoichiometries (estimated to be 1891), alternatively spliced capsid protein arrangements, packaged DNA, and payload DNA methylation states. Within this mix, it is observed that some viral particles are capable of more effectively delivering the payload DNA and having the DNA express robustly (high quality/performance rAAV particles). In contrast, an rAAV that is empty or fails to deliver its DNA payload (maybe due to suboptimal post translational modifications), or results in low gene expression, would collectively be regarded as low quality/performance rAAVs. The ratio of functional to nonfunctional particles can determine clinical safety and potency. The infectious titer can be determined by quantifying the number of rAAV virions that successfully deliver the DNA payload. This number is often much lower than the total viral titer, which is typically quantified by counting the number of viral genomes (e.g. with qPCR or ddPCR). A skilled person in the art of viral production will recognize that different rAAV designs may yield dramatically different viral titers and infectious titers, even when the manufacturing strategy is held constant.

[00248] In some embodiments, the present teachings provide methods of identifying a Synthetic Helper Gene Product (SHGP) capable of improving manufacturability of AAV particles in host cells and increase quantity and/or quality (infectiousness) of produced AAV vectors. [00249] Genetic engineering of the viral producer cell line seems to be a straightforward approach to improve properties of cell lines for viral production. However, the genetic modification linkage between cell genome and viral phenotype is lost as soon as the virus leaves the cell; therefore, it is hard to link the phenotype of a virus to the cell that produced it. This means that if a cell mutant were to be improved and were to generate more/better viruses, the viruses would provide no indication of which cell mutant from the high diversity cell pool with improved production properties they originated from. Some efforts have found solutions to this by using the virus to deliver and store genetic modifications, such as CRISPRi or siRNA. However, these approaches are limited in that they only target a small number of genes, and they only modulate expression of existing genes in the cells. Other efforts have attempted to use viruses to deliver mutations, such as by encoding repair fragments and guide RNAs, to packaging cells. However, this is challenging and suffers from only targeting a single gene at a time.

[00250] The methods and compositions provided herein have several advantages over existing approaches. In contrast to a single siRNA or sgRNA, or any other programmable gene expression modulator that will be able to modulate only one single target (determined by its base-pairing complementarity), a single Synthetic Helper Gene Product (SHGP) has the potential to interact with and modulate multiple (or even the majority of) biomolecules in a cell to varying degrees, including simultaneously targeting of multiple targets. Each SHGP may have a various degree of affinity to multiple protein targets, nucleic acids and/or small molecules. Because SHGPs exploit a post-translational approach to modulating cell behavior, they can interact with a much greater number of biomolecules inside of a cell, and the accumulated effects of these interactions and perturbations collapse into a single cellular phenotype.

[00251] In addition to merely providing for an increase or decrease in the amount of expression of a specific gene, Synthetic Helper Gene Products (SHGPs) can engage their targets in a manner that can fundamentally alter the target’s behavior in ways that were previously unattainable through conventional genetic regulation, alternative splicing, or post- translational modifications. For instance, effector proteins in bacteria, such as SopE from Salmonella, have been specifically engineered to target and manipulate host CDC42 and Rael GTPases, thus influencing cellular signaling pathways. Moreover, cholera toxin has been utilized to specifically target the Gs alpha subunit, leading to the constitutive activation of adenylate cyclase, resulting in the alteration of host cell functions. [00252] Similarly, diphtheria toxin has been found to specifically target and ADP- ribosylate elongation factor 2 (EF-2), thereby inhibiting protein synthesis within the host. In the realm of viral proteins, Tat protein in HIV targets the TAR RNA element, subsequently affecting host transcription dynamics. Hepatitis C Virus (HCV) core protein alters lipid metabolism by targeting the host microsomal triglyceride transfer protein (MTP). Helicobacter pylori CagA protein, through targeting and phosphorylation of SHP-2 phosphatase, can change host cell signaling.

[00253] Furthermore, anthrax toxin, composed of Protective Antigen (PA), Lethal Factor (LF), and Edema Factor (EF), where LF specifically targets host MAPK kinases, and EF targets adenylate cyclase, illustrates the diversity in target selection and function modulation. Botulinum neurotoxin offers an example of targeting SNARE proteins such as SNAP -25, effectively inhibiting neurotransmitter release. The proteins E6 and E7 in Human Papillomavirus (HPV) are employed to target p53 for degradation and retinoblastoma protein (Rb) to influence cell cycle regulation, respectively.

[00254] In addition, Shiga Toxin serves to target 28S rRNA of the host 60S ribosomal subunit, leading to the inhibition of protein synthesis. Specific engineered systems also include the targeting of specific genes involved in cell cycle regulation, apoptosis, and metabolism by Forkhead Box Proteins (FOXO), or the regulation of cell adhesion and migration through Src kinase targeting focal adhesion kinase (FAK).

[00255] These examples demonstrate that polypeptides are highly effective at manipulating cell behavior, having been successfully used to disrupt DNA binding activity of multiple transcription factors (as with short peptides), to target mRNA structures (as with nanobodies), or to alter intracellular trafficking (as with calreticulin). In all these cases, the new biological activity effected by the peptide or protein was not encoded by the original organism, thereby offering a novel means of achieving specific phenotypes that no amount of traditional genetic regulation could replicate. This broad array of applications and targets underscores the diverse potential of SHGPs in altering cellular behavior in precise and programmable ways.

[00256] Various SHGPs may interact with a variety of different proteins, nucleic acids or other biological targets to different degrees. They may potentially interact with several intracellular biological targets. Here are a few non-limiting examples. SHGPs can selectively inhibit the RIG-I/MDA5/0AS1 pathways, preventing ISG induction, resulting in reduced inhibition of AAV production. SHGPs can also target proteins like IFN-beta, thereby increasing AAV yield by blocking the direct antiviral effects. By inhibiting specific inflammasomes like NLRC4 and PYCARD/ASC, SHGPs can reduce inflammation, improving cell fitness and AAV production. In addition, the inhibition of the cGAS/STING pathway through SHGPs can improve AAV replication and production by reducing antiviral responses. SHGPs can also alleviate cell stress in AAV production by inhibiting protein kinase C, enhance AAV yield through the mitigation of cellular stress response by modulating MAPK activity, control glycosylation to reduce immunogenicity, and fine-tune viral assembly and transduction efficiency through ubiquitination and sumoylation management. Moreover, SHGPs can assist in proper capsid assembly, thereby reducing the formation of non-functional capsids, and prevent premature cell death by interacting with AAV replicase protein Rep78, leading to increased stability and productivity in AAV production. They may potentially interact with several intracellular biological targets. This large potential breadth of perturbation is highly desirable. For each of these interactions, in addition to simple increases or decreases in activity, allosteric modulation (a common type of post translational regulatory action) can increase the number of biological behaviors, for example altering specificity of binding proteins, stabilizing rare protein conformations to generate new enzymatic reactions, or increasing promiscuity of proteolysis. In such cases, there is not a simple increase or decrease in protein’s canonical behavior; instead, the collection of all possible protein behaviors, which might be described as a protein’s latent functional promiscuity, can be rapidly and reversibly unlocked by such post-translational perturbations from an introduced SHGP. The accumulation of multiple potential changes in activity across multiple targets greatly surpasses the breadth in functional perturbation that can be achieved by targeted methods of genetic regulation.

[00257] In some embodiments, the methods described herein utilize the concept of selfish elements (e.g., SHGP gene sequences) that can improve the efficiency of their replication in a competitive environment (e.g., a DNA library encoding SHGPs). By performing one or more rounds of selection with a library encoding SHGPs, cells with high viral manufacturing capacity can be generated. Typically, more rounds of selection would enrich cell (or viral) population with sequences that encode SHGPs that provide selective advantages for AAV vectors containing the corresponding SHGP coding sequences. Some of the described methods provide a way for SHGPs to competitively self-replicate using a viral vector; accordingly, tools of directed evolution and selective/competitive enrichment are used for the discovery of SHGPs. In some embodiments, the virus is used to harbor a selfish element that directly alters cell behavior and is easily identified. In some embodiments, the virus is used to hold the element that optimizes the cell rapidly and directly with the element also being readily identifiable using standard techniques like molecular cloning, and sequencing/NGS. Further, SHGP gene sequence libraries can be pooled, and the performance of all hits easily validated using NGS. This contrasts with drug screening approaches in which pooling an entire library of millions of compounds would make it impossible to effectively identify hits, which leads to the requirement of microwell plate and automated high throughput screening approaches. Because SHGPs are genetically encodable and can be expressed ribosomally, the DNA that encodes these highly efficient Synthetic Helper Gene Product perturbation modules can be packaged into the viral vector, providing an facile way of exploiting genetic information storage and transmission for the identification of SHGP -based viral replication enhancers. [00258] SHGPs provide many advantages over traditional cell line engineering strategies and chemical additive strategies. SHGPs are genetically programmable, selectable, and internally expressed, which both improves target selectivity and potency making them an ideal tool for general purpose, post-translational perturbation of cellular proteome.

[00259] Synthetic Helper Gene Products can target "undruggable" molecules due to their ability to interact with large, flat, and shallow binding surfaces that are difficult for small molecules to engage. Small molecules typically require well-defined pockets to bind effectively, whereas Synthetic Helper Gene Products can exploit their structural diversity and conformational flexibility to interact with challenging targets. Synthetic Helper Gene Products can interact with non-protein molecules, such as nucleic acids, lipids, and carbohydrates, giving them a distinct advantage over small molecules, which canonically target globular proteins. Synthetic Helper Gene Product structural diversity and adaptable binding modes enable them to recognize and to bind various types of molecules, providing a versatile approach for cellular perturbation. Synthetic Helper Gene Products can exhibit higher binding specificity and affinity for their target proteins compared to small molecules (one might consider the difference between antibody affinity vs small molecule affinity). SHGPs can be designed or optimized to interact with multiple targets simultaneously or sequentially (e.g. multi-specific nanobody fusions might target several intracellular targets simultaneously). SHGPs can interact with target proteins at allosteric sites, which are distinct from the active site. This mode of interaction can modulate protein function in a more nuanced and potentially reversible manner, offering an alternative approach to direct inhibition or activation of the target protein.

[00260] Synthetic Helper Gene Products can target alternatively spliced transcripts as well as overlapping protein products (as in the case of AAV Rep protein). This is particularly useful in the case of AAV, where the Rep protein (SEQ ID NO: 1-4, 10, 12, 14) encodes multiple overlapping proteins. This overlapping sequence makes it very difficult to successfully genetically engineer the rep gene sequence to selectively engineer a single protein because each desired gene mutation may have undesired results for the multiple overlapping and alternate reading frame protein products. In contrast, a Synthetic Helper Gene Product modulates its targets post translationally and can potentially selectively target a single protein species from such overlapping, alternative reading frame, or alternatively spliced genetic constructs. This provides an unusual advantage over more traditional, pure genetic engineering-based approaches.

[00261] In preferred embodiments of the disclosed methods and compositions, Synthetic Helper Gene Products are expressed in host cells from a constitutive promoter, which allows for constant, rather than inducible expression of SHGPs.

[00262] In preferred embodiments of the disclosed methods and compositions, Synthetic Helper Gene Products do not require addition of an inducer compound to mediate effect. Also, in preferred embodiments of the disclosed methods and compositions, Synthetic Helper Gene Products do not require addition of any DNA binding sequences to mediate effect. Unlike inducible systems that necessitate external compounds and/or elaborate genetic modifications for functionality, SHGPs expressed in cells inherently increase viral production through direct and autonomous interaction with existing cellular machinery, requiring no external inputs (e.g. effector compounds) or genetic alterations (e.g. a DNA binding site sequence that is naturally or engineered to be bound by the corresponding SHGP transcription factor) beyond their expression. While inducible systems, by design, control specific genes through added, synthetic regulatory elements (e.g. tet Operator sites in the case of tetR inducible transcription factors) and external inducers (e.g. tetracycline or doxycycline in the case of tetR inducible transcription factors), SHGPs expressed in host cells may enhance overall cellular output through interactions with pre-existing genomic sites (i.e. without the engineered addition of the transcription factor SHGP’s corresponding DNA binding site) and without the requirement of inducer compound addition, representing a fundamentally different approach that perturbs existing biological pathways for enhanced productivity rather than specifically controlling expression of a predefined locus. While the goal of inducible expression systems is typically to separate growth and production phases, SHGs/SHGPs may reprogram host cells during cellular growth, so decoupling of the growth and production is not needed and may even be detrimental for optimal performance of SHGPs. The addition of an inducible transcription factor protein without also including its corresponding DNA binding sequence as well as the corresponding inducer compound has not been shown to increase viral production performance (i.e. the inducible transcription factor is necessary, but not sufficient). In contrast, transcription factor based SHGPs are sufficient to drive increased viral production without needing additional DNA sequences or inducer compounds to be added.

[00263] The present teachings include a method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of a viral vector composition produced in host cells, the method comprising:

(a) culturing a first plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary for the production of the virus in the host cells; and

(iv) a Synthetic Helper Gene Product produced from a first nucleotide sequence, wherein

(v) the first nucleotide sequence is operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the first nucleotide sequence within the viral capsids or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to the one or more viral-specific packaging sequences necessary for encapsulation of the second nucleotide sequence within the viral capsids, thereby obtaining a first plurality of viral vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of viral vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for replication of the virus; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences producing the Synthetic Helper Gene Product, thereby obtaining a plurality of viral vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of viral vectors or the plurality of viral vectors of the present cycle to infect a final plurality of host cells; and (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of the viral vector composition by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from (i) the final plurality of host cells and/or (ii) a final plurality of viral vectors produced in the final plurality of host cells.

[00264] In some embodiments of the disclosed method, in step (a), the first nucleotide sequence is operably linked to the one or more viral-specific packaging sequences, and the method comprises culturing the final plurality of host cells under conditions suitable for recombinant viral production, wherein each host cell of the final plurality of host cells comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to one or more viral-specific packaging sequences and producing the Synthetic Helper Gene Product encoded by the first nucleotide sequence, thereby producing the final plurality of viral vectors from the final plurality of host cells and determining the Synthetic Helper Gene Product by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from the final plurality of viral vectors.

[00265] In some preferred embodiments of the disclosed method, the viral vector composition is an adeno-associated virus (AAV) vector composition; the at least one viral replication gene comprises at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14); the at least one viral structural gene comprises at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15); the at least one additional viral gene comprises at least one AAV helper gene (SEQ ID NO: 16-23); and the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9). In other preferred embodiments of the disclosed method, the viral vector composition is a lentivirus vector composition; the at least one viral replication gene comprises at least one lentiviral pol gene (SEQ ID NO: 25); the at least one viral structural gene comprises at least one lentiviral gag gene (SEQ ID NO: 26) and at least one env gene (SEQ ID NO: 27); the at least one additional viral gene comprises at least one lentiviral rev gene (SEQ ID NO: 24); and the one or more viral-specific packaging sequences comprise a Psi sequence (SEQ ID NO: 34). [00266] In some embodiments of the disclosed method, the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences further encodes a reporter, a therapeutic payload or a selectable marker. In some embodiments, the disclosed method further comprises step (e): generating new viral vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the viral vector composition of increased viral titer and/or transduction efficiency. In some embodiments, generating new viral vectors comprises: culturing a new plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the new plurality of host cells comprises the Synthetic Helper Gene Product determined in (d) and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and (iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload.

[00267] In some embodiments, the disclosed method produces the viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from the group consisting of viral titer and transduction efficiency. [00268] The present teachings also include a method of producing a viral vector composition of increased viral titer and/or transduction efficiency in host cells, the method comprising:

(A) obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of a viral vector composition by performing the following steps: (a) culturing a first plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a Synthetic Helper Gene Product encoded by a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the first nucleotide sequence within the viral capsids or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to the one or more viral-specific packaging sequences necessary for encapsulation of the second nucleotide sequence within the viral capsids, thereby obtaining a first plurality of viral vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps (bl)-(b2) one or more times in cycles:

(bl) allowing a plurality of viral vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for replication of the virus; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences producing the Synthetic Helper Gene Product, thereby obtaining a plurality of viral vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of viral vectors or the plurality of viral vectors of the present cycle to infect a final plurality of host cells; and

(d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of the viral vector composition by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from (i) the final plurality of host cells and/or (ii) a final plurality of viral vectors produced in the final plurality of host cells; and

(B) generating new viral vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the viral vector composition of increased viral titer and/or transduction efficiency.

[00269] The present teachings also include a method of producing a viral vector composition of increased viral titer and/or transduction efficiency in host cells, the method comprising:

(a) culturing a first plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a Synthetic Helper Gene Product encoded by a first nucleotide sequence, wherein the first nucleotide sequence is operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the first nucleotide sequence within the viral capsids, thereby obtaining a first plurality of viral vectors comprising the first nucleotide sequence from the first plurality of host cells;

(c) allowing the first plurality of viral vectors to infect a final plurality of host cells; and (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of the viral vector composition by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from the final plurality of host cells.

[00270] In some embodiments, the disclosed method further comprises, prior (c) and after (a), repeating the following steps (bl)-(b2) one or more times in cycles:

(bl) allowing a plurality of viral vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for replication of the virus; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences producing the Synthetic Helper Gene Product, thereby obtaining a plurality of viral vectors of the present cycle comprising the first nucleotide sequence; and further comprises allowing the plurality of viral vectors of the present cycle to infect a final plurality of host cells in (c), wherein one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of the viral vector composition are determined in (d) by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from a final plurality of viral vectors produced in the final plurality of host cells.

[00271] In some embodiments, the steps (bl)-(b2) of the disclosed method are repeated 2, 3, 4, 5, 10 or more times.

[00272] In some embodiments, the disclosed method produces the viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from the group consisting of viral titer and transduction efficiency. [00273] In some embodiments of the disclosed methods, the Synthetic Helper Gene Product is not essentially present in the viral vector composition of increased viral titer and/or transduction efficiency.

[00274] In some embodiments of the disclosed methods, the first plurality of host cells comprises at least 100,000 host cells each comprising structurally different Synthetic Helper Gene Products.

[00275] In some embodiments of the disclosed methods,

(i) the viral vector composition is an adeno-associated virus (AAV) vector composition;

(ii) the at least one viral replication gene comprises at least one AAV replication gene;

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene;

(iv) the at least one additional viral gene comprises at least one AAV helper gene; and

(v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs).

[00276] In some embodiments of the disclosed methods,

(i) the viral vector composition is a lentivirus vector composition;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene;

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene and at least one env gene;

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene; and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence.

[00277] In some embodiments of the disclosed methods, each host cell of first plurality of host cells and each host cell of final plurality of host cells are mammalian host cells.

[00278] In some embodiments of the disclosed methods, the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences further encodes a reporter, a therapeutic payload or a selectable marker.

[00279] In some embodiments of the disclosed methods, generating new viral vectors comprises: culturing a new plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the new plurality of host cells comprises the Synthetic Helper Gene Product determined in (d) and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and (iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload.

[00280] The present teachings also include a plurality of host cells permissive for replication of a virus, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary for the production of the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; wherein the Synthetic Helper Gene Product increases a characteristic of viral vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of viral vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of viral vectors is selected from the group consisting of viral titer and transduction efficiency.

[00281] In some preferred embodiments of the disclosed method, the virus is an adeno- associated virus (AAV); the at least one viral replication gene comprises at least one AAV replication gene; the at least one viral structural gene comprises at least one AAV capsid encoding gene; the at least one additional viral gene comprises at least one AAV helper gene; and the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9). In other preferred embodiments of the disclosed method, the virus is a lentivirus; the at least one viral replication gene comprises at least one lentiviral pol gene (SEQ ID NO: 25); the at least one viral structural gene comprises at least one lentiviral gag gene (SEQ ID NO: 26) and at least one env gene (SEQ ID NO: 27); the at least one additional viral gene comprises at least one lentiviral rev gene (SEQ ID NO: 24); and the one or more viral-specific packaging sequences comprise a Psi sequence (SEQ ID NO: 34).

[00282] In some embodiments, the plurality of host cells comprises at least 10,000 host cells. [00283] The present teachings also include a method of producing a viral vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(a) culturing a plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; and (b) producing the viral vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the viral vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral titer and/or transduction efficiency of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

[00284] In some preferred embodiments of the disclosed method, the viral vector composition is an adeno-associated virus (AAV) vector composition; the at least one viral replication gene comprises at least one AAV replication gene; the at least one viral structural gene comprises at least one AAV capsid encoding gene; the at least one additional viral gene comprises at least one AAV helper gene; and the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9). In other preferred embodiments of the disclosed method, the viral vector composition is a lentivirus vector composition; the at least one viral replication gene comprises at least one lentiviral pol gene (SEQ ID NO: 25); the at least one viral structural gene comprises at least one lentiviral gag gene (SEQ ID NO: 26) and at least one env gene (SEQ ID NO: 27); the at least one additional viral gene comprises at least one lentiviral rev gene (SEQ ID NO: 24); and the one or more viral-specific packaging sequences comprise a Psi sequence (SEQ ID NO: 34).

[00285] Disclosed herein is also a method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of adeno-associated virus (AAV) vector composition, the method comprising: [00286] (a) culturing a first plurality of host cells permissive for AAV replication under conditions suitable for recombinant AAV production, wherein each host cell of the first plurality of host cells comprises:

[00287] (i) at least one AAV replication protein produced from at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

[00288] (ii) at least one AAV capsid encoding protein produced from at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15);

[00289] (iii) at least one AAV helper protein produced from at least one AAV helper gen (SEQ ID NO: 16-23) e; and

[00290] (iv) a Synthetic Helper Gene Product produced from a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to at least two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to at least two functional AAV ITRs (SEQ ID NO: 8-9), thereby obtaining a first plurality of AAV vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

[00291] (b) optionally, repeating the following steps one or more times in cycles:

[00292] (bl) allowing a plurality of AAV vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for AAV replication; and

[00293] (b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant AAV production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the at least two functional AAV ITRs (SEQ ID NO: 8-9) producing the Synthetic Helper Gene Product, thereby obtaining a plurality of AAV vectors of the present cycle comprising the first nucleotide sequence;

[00294] (c) allowing the first plurality of AAV vectors or the plurality of AAV vectors of the present cycle to infect a final plurality of host cells; and (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of AAV vector composition by analyzing nucleotide sequences operably linked to the at least two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) from (i) the final plurality of host cells and/or (ii) a final plurality of AAV vectors produced in the final plurality of host cells. [00295] Disclosed herein is also a method of producing an adeno-associated virus (AAV) vector composition, the method comprising:

[00296] (a) culturing a first plurality of host cells permissive for AAV replication under conditions suitable for recombinant AAV production, wherein each host cell of the first plurality of host cells comprises:

[00297] (i) at least one AAV replication protein produced from at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

[00298] (ii) at least one AAV capsid encoding protein produced from at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15);

[00299] (iii) at least one AAV helper protein produced from at least one AAV helper gene (SEQ ID NO: 16-23); and

[00300] (iv) a Synthetic Helper Gene Product produced from a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to at least two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to at least two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9), thereby obtaining a first plurality of AAV vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

[00301] (b) optionally, repeating the following steps one or more times in cycles:

[00302] (bl) allowing a plurality of AAV vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for AAV replication; and [00303] (b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant AAV production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the at least two functional AAV ITRs (SEQ ID NO: 8-9) producing the Synthetic Helper Gene Product, thereby obtaining a plurality of AAV vectors of the present cycle comprising the first nucleotide sequence;

[00304] (c) allowing the first plurality of AAV vectors or the plurality of AAV vectors of the present cycle to infect a final plurality of host cells;

[00305] (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of AAV vector composition by analyzing nucleotide sequences operably linked to the at least two functional AAV inverted terminal

- n - repeats (ITRs) (SEQ ID NO: 8-9) from (i) the final plurality of host cells and/or (ii) a final plurality of AAV vectors produced in the final plurality of host cells; and (e) obtaining new AAV vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the AAV vector composition. In some preferred embodiments, the disclosed AAV vector composition is of increased viral titer and/or transduction efficiency compared to a reference AAV vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product. In some preferred embodiments of this method, it produces the AAV vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference AAV vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from viral titer and transduction efficiency. In some embodiments of this method, the Synthetic Helper Gene Product is not essentially present in the produced AAV vector composition (e.g., the Synthetic Helper Gene Product is present in host cells but is not transferred to the produced AAV vector composition).

[00306] Disclosed herein is also a plurality of host cells permissive for AAV replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one AAV replication protein produced from at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

(ii) at least one AAV capsid encoding protein produced from at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15);

(iii) at least one AAV helper protein produced from at least one AAV helper gene (SEQ ID NO: 16-23); and

(iv) a nucleotide sequence operably linked to at least two functional AAV internal terminal repeats (ITRs) (SEQ ID NO: 8-9), wherein the nucleotide sequence encodes a payload (e.g. therapeutic gene); wherein the Synthetic Helper Gene Product increases a characteristic of AAV vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of AAV vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of AAV vectors is selected from viral titer and transduction efficiency.

[00307] Disclosed herein is also a method of producing an adeno-associated virus (AAV) vector composition of increased viral titer and/or transduction efficiency, the method comprising: culturing a plurality of host cells permissive for AAV replication under conditions suitable for recombinant AAV production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one AAV replication protein produced from at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

(ii) at least one AAV capsid encoding protein produced from at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15);

(iii) at least one AAV helper protein produced from at least one AAV helper gene (SEQ ID NO: 16-23); and (iv) a nucleotide sequence operably linked between two functional AAV internal terminal repeats (ITRs) (SEQ ID NO: 8-9), wherein the nucleotide sequence encodes a payload; and (b) producing the AAV vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the AAV vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than an viral titer and/or transduction efficiency of a reference AAV vector composition produced from a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

[0078] The present teachings also include a method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of lentivirus vector composition, the method comprising:

(a) culturing a first plurality of host cells permissive for lentivirus replication under conditions suitable for recombinant lentivirus production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one lentiviral gag gene (SEQ ID NO: 26);

(ii) at least one lentiviral pol gene (SEQ ID NO: 25);

(iii) at least one lentiviral rev gene (SEQ ID NO: 24);

(iv) at least one env gene (SEQ ID NO: 27); and

(v) a Synthetic Helper Gene Product produced from a first nucleotide sequence, wherein

(vi) the first nucleotide sequence is operably linked to a Psi sequence (SEQ ID NO: 34) or (vii) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to a Psi sequence (SEQ ID NO: 34), thereby obtaining a first plurality of lentivirus vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of lentivirus vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for lentivirus replication; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant lentivirus production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iv) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the Psi sequence (SEQ ID NO: 34) producing the Synthetic Helper Gene Product, thereby obtaining a plurality of lentivirus vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of lentivirus vectors or the plurality of lentivirus vectors of the present cycle to infect a final plurality of host cells; and (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of lentivirus vector composition by analyzing nucleotide sequences operably linked to the Psi sequence (SEQ ID NO: 34) from (i) the final plurality of host cells and/or (ii) a final plurality of lentivirus vectors produced in the final plurality of host cells.

[00308] In some embodiments of the disclosed method, in step (a), the first nucleotide sequence is operably linked to the Psi sequence (SEQ ID NO: 34), and the method comprises culturing the final plurality of host cells under conditions suitable for recombinant lentivirus production, wherein each host cell of the final plurality of host cells comprises the elements (i)- (iv) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the Psi sequence (SEQ ID NO. 34) and producing the Synthetic Helper Gene Product encoded by the first nucleotide sequence, thereby producing the final plurality of lentivirus vectors from the final plurality of host cells and determining the Synthetic Helper Gene Product by analyzing nucleotide sequences operably linked to the Psi sequence from the final plurality of lentivirus vectors. In some embodiments of the disclosed method, the first nucleotide sequence operably linked to the Psi sequence further encodes a reporter, a therapeutic payload or a selectable marker. [00309] In some embodiments, the disclosed method further comprises step (e): generating new lentivirus vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the lentivirus vector composition of increased viral titer and/or transduction efficiency.

[00310] In some embodiments, the disclosed method produces the lentivirus vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference lentivirus vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from the group consisting of viral titer and transduction efficiency.

[00311] In some embodiments, the Synthetic Helper Gene Product is not essentially present in the lentivirus vector composition of increased viral titer and/or transduction efficiency.

[0079] The present teachings also include a plurality of host cells permissive for lentivirus replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one lentiviral gag gene (SEQ ID NO: 26);

(ii) at least one lentiviral pol gene (SEQ ID NO: 25);

(iii) at least one lentiviral rev gene (SEQ ID NO: 24);

(iv) at least one env gene (SEQ ID NO: 27); and

(v) a nucleotide sequence operably linked to a Psi sequence (SEQ ID NO: 34), wherein the nucleotide sequence encodes a payload; wherein the Synthetic Helper Gene Product increases a characteristic of lentivirus vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of lentivirus vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(v) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of lentivirus vectors is selected from the group consisting of viral titer and transduction efficiency.

[0080] In some embodiments, the plurality of host cells comprises at least 10,000 host cells. [0081] The present teachings also include a method of producing a lentivirus vector composition of increased viral titer and/or transduction efficiency, the method comprising: (a) culturing a plurality of host cells permissive for lentiviral replication under conditions suitable for recombinant lentiviral production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one lentiviral gag gene (SEQ ID NO: 26);

(ii) at least one lentiviral pol gene (SEQ ID NO: 25);

(iii) at least one lentiviral rev gene (SEQ ID NO: 24);

(iv) at least one env gene (SEQ ID NO: 27); and

(v) a nucleotide sequence operably linked to a Psi sequence (SEQ ID NO: 34), wherein the nucleotide sequence encodes a payload (e.g. therapeutic gene); and (b) producing the lentivirus vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the lentivirus vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral titer and/or transduction efficiency of a reference lentivirus vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(v) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

[00312] The present teachings also include a method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of recombinant adenovirus (AdV) vector composition, the method comprising: (a) culturing a first plurality of host cells permissive for AdV replication under conditions suitable for recombinant AdV production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one adenoviral capsid gene selected from the group consisting of hexon, penton base, and fiber;

(ii) at least one adenoviral core protein selected from the group consisting of protein VII and terminal protein produced from their respective genes;

(iii) at least one adenoviral early gene product selected from the group consisting of El A, E1B, E2A, and E2B produced from their respective genes (SEQ ID NO: 16-19) ; and

(iv) a Synthetic Helper Gene Product produced from a first nucleotide sequence, wherein

(v) the first nucleotide sequence is operably linked to at least two functional AdV inverted terminal repeats (ITRs) or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to at least two functional AdV ITRs, thereby obtaining a first plurality of AdV vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of AdV vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for AdV replication; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant AdV production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the at least two functional AdV ITRs producing the Synthetic Helper Gene Product, thereby obtaining a plurality of AdV vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of AdV vectors or the plurality of AdV vectors of the present cycle to infect a final plurality of host cells; and (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of AdV vector composition by analyzing nucleotide sequences operably linked to the at least two functional AdV inverted terminal repeats (ITRs) from (i) the final plurality of host cells and/or (ii) a final plurality of AdV vectors produced in the final plurality of host cells.

[00313] In some embodiments of the disclosed method, in step (a), the first nucleotide sequence is operably linked to the at least two functional AdV ITRs, and the method comprises culturing the final plurality of host cells under conditions suitable for recombinant AdV production, wherein each host cell of the final plurality of host cells comprises the elements (i)- (iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the at least two functional AdV ITRs and producing the Synthetic Helper Gene Product encoded by the first nucleotide sequence, thereby producing the final plurality of AdV vectors from the final plurality of host cells and determining the Synthetic Helper Gene Product by analyzing nucleotide sequences operably linked to the at least two functional AdV ITRs from the final plurality of AdV vectors.

[00314] The present teachings also include a plurality of host cells permissive for AdV replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one adenoviral capsid gene selected from the group consisting of hexon, penton base, and fiber; (ii) at least one adenoviral core protein selected from the group consisting of protein VII and terminal protein produced from their respective genes;

(iii) at least one adenoviral early gene product selected from the group consisting of El A, E1B, E2A, and E2B produced from their respective genes; and

(iv) a nucleotide sequence operably linked to at least two functional AdV inverted terminal repeats (ITRs), wherein the nucleotide sequence encodes a payload (e.g. therapeutic gene); wherein the Synthetic Helper Gene Product increases a characteristic of AdV vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of AdV vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of AdV vectors is selected from the group consisting of viral titer and transduction efficiency.

[00315] The present teachings also include a method of producing a recombinant adenovirus (AdV) vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(a) culturing a plurality of host cells permissive for AdV replication under conditions suitable for AdV production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one adenoviral capsid gene selected from the group consisting of hexon, penton base, and fiber;

(ii) at least one adenoviral core protein selected from the group consisting of protein VII and terminal protein produced from their respective genes;

(iii) at least one adenoviral early gene product selected from the group consisting of El A, E1B, E2A, and E2B produced from their respective genes; and

(iv) a nucleotide sequence operably linked to at least two functional AdV inverted terminal repeats (ITRs), wherein the nucleotide sequence encodes a payload (e.g. therapeutic gene); and (b) producing the AdV vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the AdV vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral titer and/or transduction efficiency of a reference AdV vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

[00316] The present teachings also include a method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of herpes simplex virus (HSV) vector composition, the method comprising:

(a) culturing a first plurality of host cells permissive for HSV replication under conditions suitable for recombinant HSV production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one HSV capsid protein selected from the group consisting of VP5, VP19C, VP23, and VP21 produced from their respective genes;

(ii) at least one HSV tegument protein selected from the group consisting of VP 16, VP 19 A, and VP22 produced from their respective genes;

(iii) at least one HSV regulatory protein selected from the group consisting of ISHGPO, ISHGP4, ISHGP22, and ISHGP27 produced from their respective genes; and

(iv) a Synthetic Helper Gene Product produced from a first nucleotide sequence, wherein

(v) the first nucleotide sequence is operably linked to at least one of TRL, IRL, IRS, TRS, or Pac sequences for packaging, or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to at least one of TRL, IRL, IRS, TRS, or Pac sequences for packaging, thereby obtaining a first plurality of HSV vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of HSV vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for HSV replication; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant HSV production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to at least one of TRL, IRL, IRS, TRS, or Pac sequences for packaging, producing the Synthetic Helper Gene Product, thereby obtaining a plurality of HSV vectors of the present cycle comprising the first nucleotide sequence; (c) allowing the first plurality of HSV vectors or the plurality of HSV vectors of the present cycle to infect a final plurality of host cells; and (d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of HSV vector composition by analyzing nucleotide sequences operably linked to the at least one of TRL, IRL, IRS, TRS, or Pac sequences from (i) the final plurality of host cells and/or (ii) a final plurality of HSV vectors produced in the final plurality of host cells.

[00317] In some embodiments of the disclosed method, in step (a), the first nucleotide sequence is operably linked to the at least one of TRL, IRL, IRS, TRS, or Pac sequences, and the method comprises culturing the final plurality of host cells under conditions suitable for recombinant HSV production, wherein each host cell of the final plurality of host cells comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the at least one of TRL, IRL, IRS, TRS, or Pac sequences, and producing the Synthetic Helper Gene Product encoded by the first nucleotide sequence, thereby producing the final plurality of HSV vectors from the final plurality of host cells and determining the Synthetic Helper Gene Product by analyzing nucleotide sequences operably linked to the at least one of TRL, IRL, IRS, TRS, or Pac sequences from the final plurality of HSV vectors.

[00318] The present teachings also include a plurality of host cells permissive for HSV replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one HSV capsid protein selected from the group consisting of VP5, VP19C, VP23, and VP21 produced from their respective genes;

(ii) at least one HSV tegument protein selected from the group consisting of VP 16, VP 19 A, and VP22 produced from their respective genes;

(iii) at least one HSV regulatory protein selected from the group consisting of ISHGPO, ISHGP4, ISHGP22, and ISHGP27 produced from their respective genes; and

(iv) a nucleotide sequence operably linked to at least one of TRL, IRL, IRS, TRS, or Pac sequences for packaging, wherein the nucleotide sequence encodes a payload (e.g. therapeutic gene); wherein the Synthetic Helper Gene Product increases a characteristic of HSV vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of HSV vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of HSV vectors is selected from the group consisting of viral titer and transduction efficiency.

[00319] The present teachings also include a method of producing a herpes simplex virus (HSV) vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(a) culturing a plurality of host cells permissive for HSV replication under conditions suitable for recombinant HSV production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one HSV capsid protein selected from the group consisting of VP5, VP19C, VP23, and VP21 produced from their respective genes;

(ii) at least one HSV tegument protein selected from the group consisting of VP 16, VP 19 A, and VP22 produced from their respective genes;

(iii) at least one HSV regulatory protein selected from the group consisting of ISHGPO, ISHGP4, ISHGP22, and ISHGP27 produced from their respective genes; and

(iv) a nucleotide sequence operably linked to at least one of TRL, IRL, IRS, TRS, or Pac sequences for packaging, wherein the nucleotide sequence encodes a payload (e.g. therapeutic gene); and (b) producing the HSV vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the HSV vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral titer and/or transduction efficiency of a reference HSV vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

[00320] Various embodiments apply equally to the aspects provided herein but will for the sake of brevity be recited only once. Thus, various of the following embodiments apply equally to aspects recited above.

[00321] Exemplary embodiments of the disclosed methods are illustrated in Figs. 1-15.

[00322] Exemplary outcomes of the methods and the materials are illustrated in Figs.

16-34.

[00323] In some embodiments, disclosed herein are Synthetic Helper Gene Products can enhance viral vector production. The Synthetic Helper Gene may have post translation modifications. [00324] The incorporation of the described Synthetic Helper Gene Product into the viral vector production process can offer a number of advantages. These may include increased vector yield, enhanced vector stability, and improved transduction efficiency into target cells, among others.

[00325] In certain embodiments, the Synthetic Helper Gene Product involved in enhancing viral vector production is comprised not only of conventional L-amino acids but can incorporate non-natural amino acids, modified peptide bonds, or non-peptide bonds. Such diversity in the peptide chain broadens the potential scope of the invention and adds versatility in its applications (e.g., enhanced proteolytic stability).

[00326] In other embodiments, the Synthetic Helper Gene Product may incorporate beta-amino acids, resulting in beta-peptides. The addition of an extra carbon in the peptide backbone can offer improved metabolic stability compared to conventional alpha-peptides. This structural variation can contribute to a higher resistance to proteolytic degradation, leading to increased peptide longevity and functional efficacy.

[00327] In some instances, the Synthetic Helper Gene Product may include non-natural amino acids. These can encompass D-amino acids, amino acids with modified side chains, or entirely synthetic amino acids. Such modifications can enhance peptide stability, improve target binding specificity, or introduce novel functionality into the peptide sequence.

[00328] Furthermore, in certain embodiments, the Synthetic Helper Gene Product may include non-amide bonds. Examples of alternative types of bonds that can be used to link amino acids or amino acid mimics include ester bonds, triazole bonds formed in click chemistry reactions, or disulfide bonds. These variations in bonding can enhance the chemical diversity of the peptide, potentially introducing unique physical or chemical properties that further improve vector production.

[00329] In some embodiments, Synthetic Helper Gene Products may be designed to selfassemble into higher order structures. Such self-assembling Synthetic Helper Gene Products can form unique geometries like nanofibers, or other three-dimensional structures. These selfassembled structures can provide an advanced scaffold for packaging, offering a more efficient spatial arrangement of components, enhancing viral assembly and potentially improving the yield and stability of vectors.

[00330] In other embodiments, Synthetic Helper Gene Products can be designed to interact specifically with components of the vector or target cells. These interactions can involve specific binding to proteins such as gag, pol, or env, leading to improved vector stability or enhanced packaging efficiency. [00331] In further embodiments, Synthetic Helper Gene Products can be incorporated into a delivery system such as nanoparticles or liposomes. These peptides can be either conjugated to the surface or encapsulated within these delivery vehicles. In the case of surface conjugation, Synthetic Helper Gene Products can provide targeting capabilities, guiding the delivery vehicle to specific cell types, or modulating its interaction with cell membranes to facilitate uptake. If encapsulated, the Synthetic Helper Gene Product can potentially be protected from degradation, extending its half-life and improving its availability for enhancing packaging.

[00332] Additionally, in some embodiments, the delivery system can also include other components, such as imaging agents for tracking the delivery and distribution of the Synthetic Helper Gene Product-enhanced vectors, or therapeutic agents that can be co-delivered with the viral vectors for synergistic therapies.

[00333] In some embodiments of the disclosed methods and compositions, the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells. [00334] In some embodiments of the disclosed methods and compositions, each host cell of the plurality of host cells is a mammalian host cell.

[00335] In some embodiments of the disclosed methods and compositions, each host cell of the plurality of host cells is an insect host cell.

[00336] In some embodiments of the disclosed methods and compositions, the payload comprises a therapeutic gene.

[00337] In some embodiments of the disclosed methods and compositions, the plurality of host cells comprises at least 10,000 host cells.

[00338] In some embodiments of the disclosed methods and compositions, the Synthetic Helper Gene Product is not essentially present in the viral vector composition of increased viral titer and/or transduction efficiency.

[00339] In some embodiments of the disclosed methods and compositions, each host cell of a plurality of host cells comprises: (i) at least one viral replication protein essential for the replication of the virus produced from at least one corresponding viral replication gene; (ii) at least one viral structural protein essential for formation of viral capsids of the virus produced from at least one corresponding viral structural gene; and (iii) at least one additional viral protein necessary for the production of the virus in the host cells, produced from at least one corresponding viral gene.

[00340] In some embodiments of the disclosed methods and compositions, each host cell of a plurality of host cells comprises: (i) at least one AAV replication protein produced from at least one corresponding AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14); (ii) at least one AAV capsid encoding protein produced from at least one corresponding AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15); and (iii) at least one AAV helper protein produced from at least one corresponding AAV helper gene (SEQ ID NO: 16-23).

[00341] In some embodiments of the disclosed methods and compositions, each host cell of a plurality of host cells comprises: (i) at least one lentiviral gag protein produced from at least one corresponding lentiviral gag gene (SEQ ID NO. 26); (ii) at least one lentiviral pol protein produced from at least one corresponding lentiviral pol gene (SEQ ID NO: 25); (iii) at least one lentiviral rev protein produced from at least one corresponding lentiviral rev gene (SEQ ID NO: 24); and (iv) at least one lentiviral env protein produced from at least one corresponding lentiviral env gene (SEQ ID NO: 27). In some embodiments, the at least one env gene is an envelope gene encoding a glycoprotein from an enveloped virus. In one particular embodiment, the at least one env gene encodes the protein comprising amino acid sequence set forth in SEQ ID NO: 27.

[00342] In some embodiments of the disclosed methods and compositions, each host cell of a plurality of host cells comprises: (i) at least one adenoviral capsid protein selected from the group consisting of hexon, penton base, and fiber produced from their respective genes; (ii) at least one adenoviral core protein selected from the group consisting of protein VII and terminal protein produced from their respective genes; and (iii) at least one adenoviral early gene product selected from the group consisting of E1A, E1B, E2A, and E2B produced from their respective genes (SEQ ID NO: 16-19).

[00343] In some embodiments of the disclosed methods and compositions, each host cell of a plurality of host cells comprises: (i) at least one HSV capsid protein selected from the group consisting of VP5, VP19C, VP23, and VP21 produced from their respective genes; (ii) at least one HSV tegument protein selected from the group consisting of VP 16, VP19A, and VP22 produced from their respective genes; and (iii) at least one HSV regulatory protein selected from the group consisting of ISHGP0, ISHGP4, ISHGP22, and ISHGP27 produced from their respective genes.

[00344] In some particular embodiments, the AAV replication gene present in each host cell of the first plurality of host cells encodes a protein that comprises an amino acid sequence having at least 90%, at least 95% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO 1-4, 10, 12, 14.

[00345] In some particular embodiments, the AAV capsid encoding gene present in each host cell of the first plurality of host cells encodes a protein that comprises an amino acid sequence having at least 90%, at least 95% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 5-7, 11, 13, 15. In some particular embodiments, the AAV helper gene present in each host cell of the first plurality of host cells encodes a protein that comprises an amino acid sequence having at least 90%, at least 95% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 16-23.

[00346] In some embodiments, after culturing a plurality of host cells permissive for AAV replication under conditions suitable for recombinant AAV production, the AAV vector composition of increased viral titer and/or transduction efficiency is produced from the plurality of host cells by methods known in the art. In preferred embodiments, the plurality of host cells permissive for AAV replication is cultured in vitro in a liquid culture medium such that host cells of the plurality of host cells produce AAV viral particles, which then are harvested from the culture medium. In some embodiments, producing AAV vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells comprises purifying the AAV viral particles from the culture medium. In some embodiments, harvested AAV viral particles comprise heterologous nucleic acid(s) encoding one or more heterologous gene products. In some embodiments, heterologous gene products comprise a polynucleotide or a polypeptide. In some embodiments, the harvested AAV viral particles are purified. In some embodiments, the AAV viral particles are purified to at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or greater than 99%, purity. Suitable liquid culture media include any culture medium that provides for growth and/or viability of a mammalian cell (if host cells are mammalian cells) or an insect cell (if host cells are insect cells) in in vitro culture.

[00347] In some embodiments, the AAV vector composition of increased viral titer and/or transduction efficiency has an infectious unit titer which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than an infectious unit titer of a reference AAV vector composition produced without Synthetic Helper Gene Products.

[00348] In some embodiments, the harvesting comprise harvesting the first, second, third, or higher order plurality of AAV capsids and infecting a murine or primate animal with the first, second, third, or higher order plurality of AAV capsids, wherein the most highly infectious AAV vectors of the AAV vector composition efficiently deliver the nucleotide sequence positioned between two ITRs (SEQ ID NO: 8-9) to animals cells, while the least infectious AAV vectors of the AAV vector composition fail to deliver or inefficiently deliver the nucleotide sequence positioned between two ITRs (SEQ ID NO: 8-9) to animals cells. [00349] Exemplary host cells suitable for the methods and the compositions provided herein include, without limitation, the following cell lines. (1) HEK293 cells and derivatives or related strains. (2) PER.C6 cells; PER.C6 cells are a human retinal pigment epithelial cell line that are also commonly used for AAV production. They are easy to grow and maintain, and they have high transfection efficiency. (3) BTI-TN-5B1-4 cells (High Five cells). These cells are derived from Trichoplusia ni (cabbage looper) ovarian cells and can also be used in the baculovirus expression vector system. High Five cells can grow to high densities in suspension culture and are known to produce high yields of recombinant proteins. (4) Sf9 insect cells. (5) Viro9 cells. (6) CHO cells and derivatives or related strains. (7) BHK cells; Baby hamster kidney (BHK) cells have also been used for AAV production, particularly for some AAV serotypes that may not be efficiently produced in other cell lines. BHK cells can be transfected with plasmids encoding the AAV vector genome, rep (SEQ ID NO: 1-4, 10, 12, 14) and cap genes (SEQ ID NO: 5-7, 11, 13, 15), and helper (SEQ ID NO: 16-23) functions from adenoviruses.

[00350] In some embodiments, the viral vectors produced by the plurality of host cells are viral vectors of a non-enveloped virus. In other embodiments, the viral vectors produced by the plurality of host cells are viral vectors of an enveloped virus.

[00351] Culture of the viral vector composition-producing host cell can be performed under known culture conditions. For example, the host cell is cultured at a temperature of 30 to 37° C., a humidity of 95%, and a CO2 concentration of 5 to 10% (v/v). However, culture conditions of the viral vector composition-producing host cell are not limited to the above- mentioned culture conditions. The cell culture may be performed at a temperature, a humidity, and a CO2 concentration out of the above-mentioned ranges, as long as desired cell growth and production of the viral vector composition are accomplished. A culture period is not particularly limited, and for example, the cell culture is continued for 12 to 150 hours, preferably 48 to 120 hours.

[00352] In some embodiments, the plurality of host cells comprising elements necessary for production of the viral vector composition also comprise a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload. In some embodiments, the payload comprises a reporter, a therapeutic moiety (e.g., protein, RNA, DNA, enzyme, growth factor, cytokine, receptor, etc.) or a selectable marker. To control expression of the payload, a suitable promoter, enhancer, terminator, and/or other transcriptional regulatory elements may be inserted into the nucleic acid encoding the payload. In some particular embodiments, the payload comprises a therapeutic payload (e.g., therapeutic gene). Non limiting examples of FDA-approved therapeutic payloads include: 1) human retinoid isomerohydrolase RPE65 (Serotype: AAV2); human survival motor neuron protein (Serotype: AAV9); and hFIX-Padua (Coagulation factor) (Serotype: AAV5). Multiple examples of viral vector compositions exist in clinical trials (see, e.g., Clinical Trials Database).

[00353] In some embodiments, TU:VG ratio of the AAV vector composition of increased viral titer and/or transduction efficiency is from 1 : 100 to 1 :50, from 1 : 50 to 1 :20, from 1 :20 to 1 : 10, from 1 : 10 to 1 :5, from 1 :5 to 1 :2, or from 1 :2 to 1 : 1.

[00354] In some embodiments, the AAV vector composition of increased viral titer and/or transduction efficiency has a viral genome titer which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral genome titer of a reference AAV vector composition produced without Synthetic Helper Gene Products.

[00355] In some embodiments, the above-described method produces AAV composition having a titer of 20 lU/cell or more, wherein a reference titer of infectious units produced without the SHGP is 10 lU/cell (at least 2-fold increase in lU/cell).

[00356] It should be noted that different AAV serotypes have different viral titers. Additionally, different packaging strategies have different viral titers.

[00357] The method works by applying competitive genetic selection on the functional attributes of rAAV vectors (e.g. infectiousness). Said rAAV vectors were assembled/packaged in a cell comprising a genetically encoded, ribosomally synthesized, post-translationally modified Synthetic Helper Gene Product. Said rAAV harbors DNA encoding a Synthetic Helper Gene Product (or indicating via barcode) that is/was present during assembly/packaging of the rAAV. The peptide-induced modulation of the host cell’s packaging environment alters the rAAV assembly in ways that dramatically alter yield, infectiousness and other rAAV attributes. The SHGP-mediated enhancements of rAAV packaging are easily coupled to the propagation of DNA molecules that encode the endogenously expressed Synthetic Helper Gene Product (or identifying DNA barcodes). After AAV packaging, a viral capsid contains DNA encoding/identifying a peptide library element. Different library members are expected to possess different functional attributes as a result of their having been assembled in the presence of the Synthetic Helper Gene Product encoded by the DNA that they harbor. These rAAVs are used to transfect naive cells. The transfer of DNAs encoding/identifying peptide library elements to naive cells is linked to the quality/infectivity of the AAV capsid, which may be positively, negatively, or neutrally impacted by the presence of the Synthetic Helper Gene Product during rAAV packaging. Synthetic Helper Gene Products that reduce rAAV packaging, yield, infectivity, or other attributes that would facilitate their propagation in a competitive enrichment assay are rapidly depleted from the library. On the other hand, Synthetic Helper Gene Products that confer large improvements to rAAV packaging, yield, infectivity, or other attributes that would facilitate their propagation in a competitive enrichment assay are strongly selected for and rAAVs harboring DNAs that encode or identify these Synthetic Helper Gene Products rapidly increase their population. In this way, we can exploit the functional attributes of rAAVs to enrich for DNAs encoding Synthetic Helper Gene Products that increase the manufacturability of rAAVs. The DNA molecules can be recovered after transfection for analysis and identification of the peptide library elements. The approach can be applied to combinations of Synthetic Helper Gene Products as well in order to identify combinations of Synthetic Helper Gene Products that synergize to further enhance the yield and quality of rAAV.

[00358] The value of this method is a large throughput advantage as an entire peptide library is selected for the ability of the library elements to positively modulate/enhance AAV manufacturability. In contrast, library elements that negatively modulate AAV manufacturability or cell viability are immediately depleted from the library and/or out- competed by performance-enhancing SHGPs. The method is intracellular; the Synthetic Helper Gene Product library is expressed endogenously. This increases the local concentration bioavailability of the peptide library elements.

[00359] The present disclosure also provides platform technologies for optimized production of AAV vectors as well as methods for identifying said optimizations. The present disclosure provides methods that use library-based approaches for identifying Synthetic Helper Gene Products, that when added to or produced by host cells during AAV production, increase manufacturability of AAVs. The present disclosure provides a novel discovery platform technology, where DNA that encodes Synthetic Helper Gene Products is expressed by host cells that are simultaneously producing AAVs, wherein DNA encoding said SHGP (or a related barcode) can be packaged into AAV capsids. The effect of the SHGP on AAV production results in the DNA encoding said SHGP (or a related barcode) being packaged more/less efficiently or into more/less infectious AAV particles. Viral vector libraries (containing SHGPs or barcodes) produced from mammalian cells can be analyzed as a pool by NGS in order to understand the functional properties conferred upon the AAV vector by the presence of the SHGP during the viral packaging in the mammalian cell. Desired characteristics include but are not limited to enhanced or improved viral vector production, infectiousness, empty :full capsid ratio, gene expression.

[00360] In some embodiments, an AAV vector is therapeutically active. However, in some embodiments, provided methods may yield non-functional AAV vectors that lack one or more functional characteristics, but retain other characteristics of interest. In some embodiments, an AAV vector is non-functional or has reduced function for a particular characteristic. For example, in some embodiments an AAV vector may have a reduced ability to transfer a payload or may not be able to transfer a payload. In some embodiments, an AAV vector may have reduced ability to kill cancer cells. In some embodiments, an AAV vector may be therapeutically inactive.

[00361] In some embodiments of the disclosed method, the first nucleotide sequence is operably linked to at least one functional AAV inverted terminal repeat (ITR) (SEQ ID NO: 8- 9) as disclosed in Samulski RJ, Berns KI, Tan M, Muzyczka N. Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2077-81. In other embodiments of the disclosed method, the first nucleotide sequence is operably linked to two or three functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9). In some embodiments, two functional AAV ITR sequences comprise human AAV1 ITRs, human AAV2 ITRs (SEQ ID NO: 8-9), human AAV3b ITRs, human AAV4 ITRs, human AAV5 ITRs, human AAV6 ITRs, human AAV7 ITRs, human AAV8 ITRs, human AAV9 ITRs, human AAV 10 ITRs, human AAV11 ITRs, human AAV 12 ITRs, or human AAV13 ITRs. In other embodiments, two functional AAV ITR sequences comprise bovine AAV (b-AAV) ITRs, canine AAV (CAAV) ITRs, mouse AAV 1 ITRs, caprine AAV ITRs, rat AAV ITRs, or avian AAV (AAAV) ITRs

[00362] In some embodiments, the first nucleotide sequence may encode a Synthetic Helper Gene Product, a barcode that identifies the Synthetic Helper Gene Product, or both Synthetic Helper Gene Product and barcode.

[00363] In some embodiments of the disclosed method, the nucleotide sequence positioned between two inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) comprises a Synthetic Helper Gene sequence that encodes a Synthetic Helper Gene Product during rAAV production in the host cell. In other embodiments of the disclosed method, the nucleotide sequence positioned between two inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) comprise a barcode sequence that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, while a sequence that encodes a a Synthetic Helper Gene Product is present outside two ITRs (SEQ ID NO: 8-9). The latter embodiments still allow for selection of AAV-enhancing Synthetic Helper Gene Products, since after an infection round, enriched AAV vectors may be purified, and corresponding barcode sequences can be isolated and sequenced, thus decoding AAV-enhancing Synthetic Helper Gene Products.

[00364] Properties of Synthetic Helper Genes and Gene Products.

[00365] In some embodiments, the synthetic helper gene encodes an engineered binding protein. In further embodiments, the engineered binding protein is an antibody-like protein. In yet further embodiments, the antibody-like protein is selected from the group consisting of single domain antibodies and antibody mimetics. In preferred embodiments, the synthetic helper gene encodes a single domain antibody comprising at least 1 complementaritydetermining region (CDR) and preferably at least 3 CDRs. In the most preferred embodiments, the synthetic helper gene encodes a nanobody in which at least one of the CDRs has been mutated, wherein the mutations preferably occurs in CDR3. The mutation may be selected from the group consisting of substitutions, deletions, and insertions, and is designed to alter the binding specificity, affinity, or both, of the nanobody towards one or more target molecules present in human cells engineered to produce viral vectors production.

[00366] In some embodiments, antibody -like synthetic helper genes may be derived from engineered variants of various antibody mimetic proteins. These antibody mimetic proteins include, but are not limited to, affibodies, designed ankyrin repeat proteins (DARPins), monobodies, anticalins, affimers, alphabodies, and centyrins. In preferred embodiments, the antibody mimetic protein is engineered to have a binding interface comprising between 5 and 40 specificity-determining residues. In further preferred embodiments, the specificity-determining residues are located on surface-exposed regions of the protein and are arranged to form a binding pocket or interface that complements the structure of a target molecule involved in viral vector production pathways.

[00367] In some embodiments, the synthetic helper gene encodes an engineered binding protein. In further embodiments, the engineered binding protein is an engineered transcription factor or transcription factor like protein (i.e. Al-designed proteins). In preferred embodiments, the engineered transcription factor comprises a nuclear localization signal (NLS) to ensure efficient translocation to the nucleus of the packaging cell. In especially preferred embodiments, the transcription factor comprises one or more DNA binding motifs selected from the group consisting of helix-tum-helix (HTH), zinc finger, leucine zipper (bZIP), helixloop-helix (HLH), and homeodomain motifs.

[00368] In further embodiments of the engineered transcription factors, the synthetic helper gene is derived from a toxin-antitoxin system. In preferred embodiments, the transcription factor portion of the toxin-antitoxin system is modified to alter its DNA binding specificity, its interaction with other transcriptional regulators, or both. In especially preferred embodiments, the modified toxin-antitoxin-derived transcription factor is engineered to regulate genes involved in cellular processes that enhance viral vector production, such as cell cycle regulation, apoptosis suppression, or metabolic pathway modulation.

[00369] In some embodiments, the synthetic helper gene encodes an antibody -like protein. In some embodiments, the antibody-like protein is an antibody mimetic.

In some embodiments, the antibody-like protein is a single-domain antibody.

[00370] In some embodiments, the synthetic helper gene encodes a transcription factorlike protein. In some embodiments, the transcription factor-like protein is derived from other DNA-binding protein families.

[00371] In some embodiments, the transcription factor-like protein is derived from a toxin-antitoxin system.

[00372] In some embodiments, the antibody-like protein is a single domain antibody selected from the group consisting of nanobodies, single-chain variable fragments (scFvs), and heavy-chain only antibodies. In some embodiments, the antibody-like protein is an antibody mimetic selected from the group consisting of Affibody, Affilins, Affimers (Adhirons), Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Gastrobodies, Kunitz Domain Peptides, Monobodies, nanoCLAMPs, Repebodies, Pronectin, Centyrins, Obodies, Nanofitins, Peptibodies, Adnectins, Knottins (Inhibitor Cystine Knots).

[00373] In some embodiments, the synthetic helper gene encodes a transcription factor. In some embodiments, the transcription factor is derived from a toxin-antitoxin system, such as, for example, RelB, Phd, CcdA, ParD, HigA, HipB, DinJ, MazE, VapB, YefM, MqsA, Xre, Omega, PemI, Fic, PaaR2, AxnR, Axe, LsoA, AbiEii.

[00374] In some embodiments, SHGPs are generated from one of the following polypeptides:

1) RelB (Various Bacteria): as part of the RelBE toxin-antitoxin system, RelB acts as a transcriptional repressor that binds to DNA to regulate the expression of the operon, controlling its own expression along with the toxin RelE.

2) Phd (E. coli): In the Phd/Doc system, Phd functions as an antitoxin and a transcriptional repressor, binding to the promoter region of the operon to control the expression of both components.

3) CcdA (E. coli): Within the CcdAB system, CcdA is a transcriptional repressor that binds directly to DNA to control the operon expression, ensuring the regulation of toxin CcdB. 4) ParD (E. coli and other bacteria): In the ParDE system, ParD acts as an antitoxin that also functions as a DNA-binding protein, regulating the transcription of the toxin-antitoxin genes.

5) HigA (Pseudomonas aeruginosa and other bacteria): Part of the HigBA system, HigA binds to the operon’s promoter region as a transcriptional repressor, controlling both toxin and antitoxin gene expression.

6) HipB (E. coli): Although primarily part of the HipBA system as an antitoxin, HipB can also bind to DNA and act as a transcriptional regulator, particularly controlling the stress- induced expression of Hip A toxin.

7) DinJ (E. coli): In the DinJ-YafQ system, DinJ acts as a transcriptional repressor, binding to the promoter region to regulate the expression of the toxin YafQ and itself.

8) MazE (E. coli): In the MazEF system, it functions as a transcriptional repressor, binding DNA to control the operon’s expression.

9) VapB (Mycobacterium tuberculosis): A part of the VapBC system, it binds to DNA as a transcriptional repressor.

10) YefM (E. coli): Acts as a transcriptional regulator in the Txe/YefM system, binding DNA to regulate gene expression.

11) MqsA (E. coli): A DNA-binding protein in the MqsRA system, acting as a transcriptional regulator.

12) Xre (Various bacteriophages and bacteria): A common DNA-binding protein in phage- related toxin-antitoxin systems, often regulating lysogenic cycles.

13) Omega (E. coli and other bacteria): In the Omega-Epsilon-Zeta system, it binds to DNA and regulates transcription of the operon.

14) PemI (E. coli): Regulates the PemIK system by binding to DNA as a transcriptional repressor.

15) Fic (E. coli): As part of the Fic/Rel system, it binds to the operon’s promoter to regulate transcription.

16) PaaR2 (Pseudomonas putida): Functions as a transcriptional regulator within the PaaR2-PaaA2 system, regulating expression by binding to DNA.

17) AxnR (Various plasmids): In the AxnR-AxnA system, AxnR acts as a transcriptional regulator by binding to specific DNA sequences.

18) Axe (Plasmid-based systems): Functions as a transcriptional regulator in the Axe-Txe system, controlling gene expression by DNA binding. 19) LsoA (Lactobacillus spp.): In the LsoA-LsoB system, it acts as a transcriptional regulator that controls operon expression by binding to DNA

20) AbiEii (Lactococcus lactis): Acts as a transcriptional regulator within the AbiEi-AbiEii system, binding to DNA to control transcription.

[00375] In some embodiments, the protein encoded by the synthetic helper gene contains specificity-determining residues.

[00376] In some embodiments where the protein is a single domain antibody, the specificity-determining residues are arranged in complementarity-determining region (CDR)- like configurations.

[00377] In some embodiments where the protein is an antibody mimetic, the specificitydetermining residues are arranged in CDR-like configurations or arranged across a binding interface.

[00378] In some embodiments where the protein is a transcription factor, the specificitydetermining residues are arranged in and around DNA-binding domain (DBD) motifs.

[00379] In some embodiments, the protein encoded by the synthetic helper gene contains between 5 and 40 specificity-determining residues.

[00380] In some embodiments, the specificity-determining residues are located on surface-exposed regions of the protein.

[00381] In some embodiments, SHGP is ribosomally expressed in a packaging cell. In some embodiments, SHGP is expressed intracellularly during viral production. In some embodiments, SHGP is not incorporated into or essential present in the final viral product. In some embodiments, SHGP is not secreted from the packaging cell.

[00382] In some embodiments, SHGP manipulates cellular behavior during viral packaging. In some embodiments, SHGP may bind to various molecular targets (e.g. protein or DNA targets), wherein this binding modulates cellular processes. In some embodiments, this binding drives differential cellular phenotypes, and some of these differential cellular phenotypes result in enhanced viral vector production.

[00383] In some embodiments, SHGP comprises between 6 and 300 amino acid residues. In some embodiments, SHGP comprises between 30 and 300 amino acid residues. In some embodiments, SHGP comprises between 6 and 30 amino acid residues. In some embodiments, SHGP comprises between 6 and 100 amino acid residues. In some embodiments, SHGP comprises between 100 and 300 amino acid residues. In preferred embodiments, the size range of SHGP is determined by the starting scaffold used to select optimal SHGP in the described screening process (see Figures). In some embodiments, amino acid sequence of SHGP has less than 20, 30, 40, 50, 60, 70, 80 or 90% sequence identity to any naturally occurring protein of a host cell used for obtaining the viral vector composition capable of increasing viral titer and/or transduction efficiency. In some embodiments, amino acid sequence of SHGP has less than 20, 30, 40, 50, 60, 70, 80 or 90% sequence identity to any human virus protein.

[00384] In some embodiments, SHGP is encoded by a single DNA sequence. In some embodiments, the encoding DNA sequence has no overlapping expression products. In some embodiments, the encoding DNA sequence has no alternate splicing.

[00385] In some embodiments, SHGP is expressed in human cells that are configured to produce AAV and are actively packaging AAV. In some embodiments, SHGP improves cell behavior in such a way that AAV production is enhanced. In preferred embodiments, SHGP is present in the packaging cell during viral packaging but is absent in the final viral product formulation. In some embodiments, SHGP improves viral vector yield. In some embodiments, SHGP enhances viral vector quality.

[00386] In some embodiments, SHGPs are antibody-like proteins which include single domain antibodies and antibody mimetics. An “Antibody-like protein” is a protein that binds to a specific target molecule with high affinity and specificity, similar to antibodies, but may have a different structure. This category includes single domain antibodies and antibody mimetics. [00387] An "antibody mimetic" refers to a type of polypeptide molecule that can bind to a cognate target molecule (such as intracellular protein) with high specificity and affinity, similar to antibodies, but are not structurally identical to antibodies. Antibody mimetic is a protein that is not produced by B cells of a mammal either naturally or following immunization. Antibody mimetics are engineered proteins that are often smaller and more stable than antibodies. Like antibodies, antibody mimetics are designed to bind specifically and tightly to target molecules (“antigens”), including proteins, small molecules, or other biomolecules inside host cells.

[00388] A “Single domain antibody” is a type of antibody-like protein consisting of a single monomeric variable antibody domain, capable of binding to a specific antigen without requiring the full structure of a conventional antibody. Examples include, but are not limited to nanobodies (e.g. from camels and sharks) or scFvs.

[00389] In some embodiments, SHGPs are engineered transcription factors, which have DNA binding activity. In some embodiments, SHGPs comprise one of the following features: a. Helix-Turn-Helix (HTH): Characterized by two a-helices connected by a short sequence, with the second helix involved in direct DNA interaction. b. Zinc Finger: Comprising a zinc ion coordinated by cysteine and/or histidine residues, forming a structure that binds to the major groove of DNA. c. Leucine Zipper (bZIP): Includes a basic region that interacts with DNA and a leucine zipper for dimerization, essential for binding specificity and stability. d. Helix-Loop-Helix (HLH): Consists of two a-helices separated by a loop, with functions in both DNA binding and protein dimerization. e. Homeodomain: Featuring three a-helices, particularly the recognition helix, which is crucial for specific DNA interactions.

[00390] In some embodiments of the disclosed methods and compositions, SHGPs comprise a nuclear localization tag.

[00391] In some embodiments, the Synthetic Helper Gene Product produced during production in the host cell contains more than 10 and less than 1000 amino acid residues. [00392] In some embodiments, the Synthetic Helper Gene Product is derived from a single transcriptional and translational element.

[00393] In some embodiments of the disclosed methods and compositions, SHGP is nanobody derived from camelid heavy-chain antibodies. In some embodiments of the disclosed methods and compositions, SHGP is a single-chain variable fragment (scFv) antibody mimetic (a fusion of VH and VL domains of natural antibodies).

[00394] In some embodiments, the Synthetic Helper Gene Product is derived from a nanobody, scFv, antibody fragment, or other binder proteins.

[00395] In some embodiments, the Synthetic Helper Gene Product may be derived from one of several sources. For example, Affibody molecules may be derived from the Z domain of Protein A, Affilins from Gamma-B crystallin Ubiquitin, Affimers (Adhirons) from Cystatin, and Affitins from Sac7d (from Sulfolobus acidocaldarius). Alphabodies may be derived from Triple helix coiled coil, Anticalins from Lipocalins, Avimers from A domains of various membrane receptors, and DARPins from Ankyrin repeat motif. Additionally, Fynomers may be derived from the SH3 domain of Fyn, Gastrobodies from Kunitz-type soybean trypsin inhibitor, and Kunitz domain peptides from Kunitz domains of various protease inhibitors. Monobodies may be derived from the 10th type III domain of fibronectin, nanoCLAMPs from Carbohydrate Binding Module 32-2 (Clostridium perfringens NagH), and Optimers from a flexible nucleic acid-based scaffold, specifically a G-quadruplex structure. Further, Repebodies may be derived from leucine-rich repeats, Pronectin from the fourteenth fibronectin type-III scaffold of Human Fibronectin (14Fn3), Centyrins from highly stable fibronectin type III (FN3) domain, and Obodies may be a high-affinity binding protein domain specifically engineered to bind to Hen Egg-white Lysozyme. These embodiments represent various forms of the Synthetic Helper Gene Product and are intended to be illustrative rather than exhaustive. Other variations and modifications are possible within the scope of the invention as defined by the appended claims.

[00396] In some embodiments, the Synthetic Helper Gene Product is derived from a transcription factor.

[00397] In some embodiments, the Synthetic Helper Gene Product is derived from a transcription factor. Additional content includes various types of transcription factors from which the Synthetic Helper Gene Product may be derived. These include, but are not limited to, Basic Leucine Zipper (bZIP) Transcription Factors, Basic Helix-Loop-Helix (bHLH) Transcription Factors, Zinc Finger Transcription Factors (such as C2H2, C2HC, C4, C6, C8 zinc fingers, among others), Helix-Turn-Helix Transcription Factors (including the homeodomain family), Winged Helix Transcription Factors (also known as forkhead box or FOX transcription factors), High Mobility Group (HMG) Transcription Factors (including the SOX proteins), Nuclear Receptors, ETS Transcription Factors, T-box Transcription Factors, Rel Homology Domain (RHD) Transcription Factors (including NF-KB or Nuclear Factor Kappa B), SMAD Transcription Factors (characterized by MH1 and MH2 domains), and MADS box Transcription Factors. These variations encompass a diverse range of transcription factors that the Synthetic Helper Gene Product may be derived from, and other variations and modifications are possible within the scope of the invention, as defined by the appended claims.

[00398] In some embodiments, the Synthetic Helper Gene Product is derived from a microbial transcription factor. Various types of transcription factors may be use, including Sigma Factors, which are prevalent in bacteria and are responsible for initiating transcription by RNA polymerase at specific promoter sequences, playing a crucial role in regulating gene expression during different stages of bacterial growth and stress responses. The MerR Family of bacterial transcription factors regulates responses to heavy metals, such as mercury and copper, and often act as sensors for metal concentrations in the environment, inducing or repressing the expression of metal resistance genes. AraC Family transcription factors are found in bacteria and regulate the utilization of arabinose as a carbon source, acting as switches and controlling the expression of multiple operons. LysR Family transcription factors, widespread in bacteria, regulate diverse biological processes, including amino acid metabolism, stress responses, and virulence. CRP (Cyclic AMP Receptor Protein) is a global transcriptional regulator in bacteria, responding to changes in cyclic AMP levels influenced by nutrient availability and other environmental factors. Fur (Ferric Uptake Regulator) controls iron homeostasis by regulating the expression of iron uptake and storage genes. NtrC Family transcription factors are involved in nitrogen metabolism, regulating the expression of genes required for nitrogen utilization. LexA Family controls the SOS response, a DNA damage repair system, by regulating the expression of genes involved in DNA repair and recombination. TetR Family transcription factors regulate antibiotic resistance by controlling the expression of efflux pumps and other resistance mechanisms, and PhoB Family transcription factors are involved in the regulation of phosphate metabolism in bacteria, controlling the expression of phosphate uptake and utilization genes. These variations encompass a diverse range of transcription factors from which the Synthetic Helper Gene Product may be derived, and other variations and modifications are possible within the scope of the invention, as defined by the appended claims.

[00399] In some embodiments, the Synthetic Helper Gene Product was selected from a large genetically encoded library having a large diversity, such as more than 1,000,000 structurally different peptides.

[00400] In some embodiments, the Synthetic Helper Gene Product is generated endogenously by the host cell through ribosomal synthesis and post-translational modification. [00401] In some embodiments, the Synthetic Helper Gene Product is generated synthetically, is supplied exogenously to the cells (e.g. mixed with a carrier/excipient/delivery vehicle e.g. liposomal delivery) and enters the host cell in order to create the cell comprising a Synthetic Helper Gene Product with enhanced rAAV production.

[00402] In some embodiments, the Synthetic Helper Gene Product is present throughout the entire rAAV production process (e.g. in the case of endogenously produced, Synthetic Helper Gene Products)

[00403] In some embodiments, the Synthetic Helper Gene Product is inducibly expressed in cells before or during the rAAV production process in order to create cells comprising a Synthetic Helper Gene Product.

[00404] In some embodiments, cells comprising Synthetic Helper Gene Products further comprise both endogenously produced Synthetic Helper Gene Products and exogenously supplied Synthetic Helper Gene Products.

[00405] In some embodiments, cells comprising Synthetic Helper Gene Products further comprise combinations of 2, 3, 4, 5, or more different Synthetic Helper Gene Products that synergize in order to further increase yield and quality of rAAV produced by the host cell. [00406] In some embodiments of the disclosed methods and compositions, the Synthetic Helper Gene Product comprises an antibody, antibody fragment or antibody mimetic.

[00407] In some embodiments of the disclosed methods and compositions, the Synthetic Helper Gene Product comprises one or more of the following: Antibodies, Nanobodies, SingleDomain Antibodies, Single-Chain Variable Fragments, Bispecific antibodies, Trispecific antibodies, Fab fragments, Fv Fragments.

[00408] In some embodiments of the disclosed methods and compositions, the Synthetic Helper Gene Product comprises one or more of the following: Affibody molecules from the Z domain of Protein A, Affilins from Gamma-B crystallin Ubiquitin, Affimers (Adhirons) from Cystatin, Affitins from Sac7d (from Sulfolobus acidocaldarius), Alphabodies from Triple helix coiled coil, Anticalins from Lipocalins, Avimers from A domains of various membrane receptors, DARPins from Ankyrin repeat motif, Fynomers from the SH3 domain of Fyn, Gastrobodies from Kunitz-type soybean trypsin inhibitor, Kunitz domain peptides from Kunitz domains of various protease inhibitors, Monobodies from the 10th type III domain of fibronectin, nanoCLAMPs from Carbohydrate Binding Module 32-2 (Clostridium perfringens NagH), Optimers from a G-quadruplex structure, Repebodies from leucine-rich repeats, Pronectin from the fourteenth fibronectin type-III scaffold of Human Fibronectin (14Fn3), Centyrins from highly stable fibronectin type III (FN3) domain, and Obodies specifically engineered to bind to Hen Egg-white Lysozyme.

[00409] In some embodiments of the disclosed methods and compositions, the Synthetic Helper Gene Product comprises an affinity purification tag appended at the N-terminus, C- terminus, or an internal site within the protein. In some embodiments, the affinity purification tag is selected from one or more of the following. 1) Peptide Tags: ALFA-tag, AviTag, C-tag, Calmodulin-tag, iCapTag™, polyglutamate tag, polyarginine tag, E-tag, FLAG-tag, HA-tag, His-tag, Gly-His-tags, Myc-tag, NE-tag, RholD4-tag, S-tag, SBP-tag, Softag 1, Softag 3, Spottag, Strep-tag, T7-tag, TC tag, Ty tag, V5 tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, SnoopTagJr, DogTag, SdyTag. 2) Covalent Peptide Tags: Isopeptag, SpyTag, SnoopTag, DogTag. 3) Protein Tags: BCCP, Glutathione-S-transferase-tag, Green fluorescent protein-tag, HaloTag, SNAP -tag, CLIP -tag, HUH-tag, Maltose binding protein-tag, Nus-tag, Thioredoxin-tag, Fc-tag, Designed Intrinsically Disordered tags, Carbohydrate Recognition Domain or CRDSAT-tag.

[00410] In some embodiments of the disclosed methods and compositions, the Synthetic Helper Gene Product comprises a transcription factor. Some of the transcription factors that can be used include one or more of the following superclasses, classes, or families. 1. Superclass: Basic Domains

- Class: Leucine zipper factors (bZIP), Helix-loop-helix factors (bHLH), Helix-loop-helix / leucine zipper factors (bHLH-ZIP), NF-1, RF-X, bHSH, or any subfamily thereof;

2. Superclass: Zinc-coordinating DNA-binding domains

- Class: Cys4 zinc finger of nuclear receptor type, diverse Cys4 zinc fingers, Cys2His2 zinc finger domain, Cys6 cysteine-zinc cluster, Zinc fingers of alternating composition, or any subfamily thereof;

3. Superclass: Helix-tum-helix

- Class: Homeo domain, Paired box, Fork head / winged helix, Heat Shock Factors, Tryptophan clusters, TEA domain, or any subfamily thereof;

4. Superclass: beta-Scaffold Factors with Minor Groove Contacts

- Class: RHR, STAT, p53, MADS box, beta-Barrel alpha-helix transcription factors, TATA binding proteins, HMG-box, Heteromeric CCAAT factors, Grainyhead, Cold-shock domain factors, Runt, or any subfamily thereof.

[00411] In some embodiments, the nucleic acid construct further comprises a regulatory element operably linked to the coding sequence, and wherein said transcription factor modulates the expression of a target gene in a host cell.

[00412] In some embodiments, the disclosed method produces the AAV vector composition of increased viral titer and/or transduction efficiency having a titer of infectious particles per cell, which is at least 20%, 40%, 60%, 80%, 100%, 200%, or 500% higher than a titer of a reference AAV vector composition, which is produced by the same procedure and the same pluralities of host cells, except for host cells used to produce the reference AAV vector composition do not comprise the nucleotide sequence that encodes the polypeptide configured to produce a Synthetic Helper Gene Product.

[00413] In some embodiments, the first plurality of host cells at step (b) comprises at least 10,000 host cells that produce different Synthetic Helper Gene Products (e.g. for a Synthetic Helper Gene Product library with diversity of about 10,000). In some embodiments, the first plurality of host cells at step (b) comprises at least a one hundred thousand (100,000) host cells that produce different Synthetic Helper Gene Products (e.g. for a Synthetic Helper Gene Product library with diversity of about 100,000). In other embodiments, the first plurality of host cells at step (b) comprises at least one million (1,000,000) host cells that produce different Synthetic Helper Gene Products (e.g. for a Synthetic Helper Gene Product library with diversity of about 1,000,000). In other embodiments, the first plurality of host cells at step (b) comprises at least ten million (10,000,000) host cells that produce different Synthetic Helper Gene Products (e.g. for a Synthetic Helper Gene Product library with diversity of about 10,000,000).

[00414] In some embodiments, AAV genome may be split in a host cell, which means that one ITR (SEQ ID NO: 8-9) is integrated into genome of the host cell and another ITR (SEQ ID NO: 8-9) is on a plasmid and functionally connected with a reporter protein, a therapeutic payload, a selectable marker or a nucleotide sequence that encodes a Synthetic Helper Gene Product. By integrating the plasmid into the correct genomic locus, one can generate a functional nucleotide sequence that is flanked by two ITRs (SEQ ID NO: 8-9), which can be further utilized, for example, during AAV packaging and/or payload production. [00415] In some embodiments, the AAV vector composition of increased viral titer and/or transduction efficiency produced by the methods disclosed herein, has one or more useful properties, including: enhanced infectiousness (greater number of rAAV particles are transduction competent); enhanced payload expression (average level of gene expression per transduction event is higher); more optimal full: empty ratio (more full capsids and fewer empty capsids); higher viral genome titer; and/or lower level of immunogenicity.

[00416] In some embodiments, the AAV vector composition of increased viral titer and/or transduction efficiency produced by the methods disclosed herein comprises one or more improved features, wherein one or more improved features comprise altered ability to transfer viral nucleic acid, altered AAV therapeutic activity, and/or decreased in percentage of the AAV population that are nonfunctional, and/or increase in the percentage of viral vector under a manufacturing practice that contain all and/or the essential nucleic acid sequences and/or other elements for their intended application.

[00417] In some embodiments, the first plurality of recombinant AAV is used to infect an animal model such as a mouse or rat, so that AAVs harboring SHGPs or barcodes that confer enhanced AAV manufacturability are transduced into the cells of the animal and maintained at a higher level compared to SHGPs/barcodes that confer no in vivo transduction enhancements.

[00418] In some embodiments, the mechanism of action of the Synthetic Helper Gene Product can be inferred by various approaches including molecular docking (e.g. reverse docking of the Synthetic Helper Gene Product to host and viral proteins), single cell RNA seq (i.e. to observe transcriptional consequences), machine learning prediction, mutational analysis, biochemical analysis of the rAAV capsid (e.g. gc/ms to determine capsid protein stoichiometry or post translational modification states), biochemical analysis of the rAAV DNA payload (e.g. methylation sequencing), quantitative microscopy/image-based profiling (e.g. in order to determine altered biological processes).

[00419] In some embodiments, next generation sequencing (NGS) is used to observe enrichment of different SHGPs, allowing one skilled in the art to infer the relative increase in AAV packaging fitness conferred by a given SHGP.

[00420] In some embodiments, two or more SHGPs in a single cell can yield synergistic effects.

[00421] In some embodiments, the SHGP library is targeted to a specific cell organelle (e.g. endoplasmic reticulum) or cell process (e.g. degradation of remaining SHGP).

[00422] In some embodiments, additional functional properties of rAAV capsids can be enriched, including enhanced stability (e.g. by performing multiple freeze thaw cycles, exposure to elevated temperatures, exposure to various pH levels, exposure to neutralizing antibodies) on the libraries before subsequent rounds of reinfection and enrichment. In some embodiments, a random mutagenesis technique may be employed, including error prone PCR, chemical mutagenesis, radiation-induced mutagenesis, or mutator enzymes like error prone polymerases.

[00423] In some embodiments, the Synthetic Helper Gene Product diversity is generated with an NNK codon, an NNS codon, an NNN codon, or other degenerate codons that allow control over the amino acid composition of a particular residue position in the Synthetic Helper Gene Product.

[00424] In some embodiments, rAAVs may be harvested from cells (e.g. centrifugation of cells and extracting rAAV from pellet), from the media (i.e. to enrich rAAVs that were secreted into media or released by lysis), from fractions of density gradient centrifugation or capillary electrophoresis (i.e. to enrich for rAAVs that have properly packaged genomes). [00425] In several embodiments of the disclosed methods, the provided host cells may include one or more Synthetic Helper Gene Products (SHGP). These SHGPs are highly versatile and may adopt a wide variety of forms. Notably, two classes of these SHGPs have been found to be particularly effective for the manipulation of cellular behavior, with the goal of optimizing viral production.

[00426] The first class is comprised of antibody-like proteins. Within this category are two subclasses: antibody mimetics and single domain antibodies. This category of SHGPs can include, but is not limited to, various types of antibody mimetics and/or single domain antibodies including nanobodies, scFvs, and other antibody-like proteins. [00427] The aforementioned antibody -mimetics proteins can be derived from a variety of sources. Affibody molecules, for example, are derivatives of the Z domain of Protein A. Affilins are drawn from Gamma-B crystallin Ubiquitin, while Affimers, also known as Adhirons, are derived from Cystatin. Affitins are taken from Sac7d, originating from Sulfolobus acidocaldarius.

[00428] Further examples of sources for antibody mimetics include, but are not limited to: Alphabodies, which are derived from Triple helix coiled coil; Anticalins, which are derived from Lipocalins; Avimers, which are sourced from A domains of various membrane receptors; DARPins, which are derived from the Ankyrin repeat motif; Fynomers, which are drawn from the SH3 domain of Fyn; Gastrobodies, which are derived from Kunitz-type soybean trypsin inhibitor; and Kunitz domain peptides, which are derived from Kunitz domains of various protease inhibitors.

[00429] In addition, Monobodies may be derived from the 10th type III domain of fibronectin, while nanoCLAMPs can be sourced from Carbohydrate Binding Module 32-2 of Clostridium perfringens NagH. Optimers may be derived from a flexible nucleic acid-based scaffold; G-quadruplex, and Repebodies are obtained from leucine-rich repeats. Pronectin is drawn from the fourteenth fibronectin type-III scaffold of Human Fibronectin (14Fn3), while Centyrins are derived from highly stable fibronectin type III (FN3) domains. Finally, Obodies are a high-affinity binding protein domain engineered to bind to Hen Egg-white Lysozyme.

[00430] At a minimal level, Synthetic Helper Genes may be described as comprising of a synthetic protein sequence engineered expressed in a cell configured to produce virus that is also actively producing viral material. The protein sequence may be one of two classes: engineered transcription factors or antibody-like proteins, in order to direct cellular perturbations to the two principle biological polymers within the cell (DNA and proteins, respectively). This core structure can be optionally linked to one or more secondary domains, which can confer additional properties to the Synthetic Helper Gene.

[00431] In some embodiments, these secondary domains may enhance or be required for the Synthetic Helper Gene's functionality in multiple ways. NLS domains must be incorporated into transcription factors to ensure the proteins entry into the nucleus where it can mediate its biological / transcriptional perturbation.

[00432] Effector domains may be incorporated into Synthetic Helper Genes, providing additional functionality beyond simple binding and inhibition. Examples of effector domains that may be included in the Synthetic Helper Genes include, but are not limited to: Ubiquitinases, Deubiquitinases, Kinase domains, Phosphatase domains, DNA-binding domains, and nuclear localization signals (NLS).

[00433] By employing these diverse sequence tags (e.g NLSs) and/or effector domains in the Synthetic Helper Genes, researchers are granted the ability to tailor the Synthetic Helper Genes to specific cellular contexts, thus opening new avenues for research in synthetic biology and gene regulation studies.

[00434] In some embodiments, provided herein are mammalian host cells and/or mammalian host cell populations that comprise a plurality of synthetic sequences comprising at least one SHG library variant and at least one identifier (barcode), and wherein the at least one identifier is positioned between the two AAV ITR (SEQ ID NO: 8-9) sequences, and where the at least one library variant is positioned outside the two AAV ITR (SEQ ID NO: 8-9) sequences. In some embodiments, provided mammalian host cells and/or mammalian host cell populations comprise a plurality of synthetic sequences comprising at least one library variant, at least one identifier (barcode), and at least one payload, where the at least one identifier and the at least one payload are positioned between the two AAV ITR (SEQ ID NO: 8-9) sequences, and where the at least one library variant is positioned outside the two AAV ITR (SEQ ID NO: 8-9) sequences. In some embodiments, provided library constructs comprise: at least one library variant, at least one identifier (barcode), and at least one payload, where the at least one library variant, the at least one identifier, and the at least one payload are positioned between the two AAV ITR (SEQ ID NO: 8-9) sequences.

[00435] Provided library constructs can be introduced into host cells using any appropriate method known in the art. In some embodiments, a library construct is introduced into a host cell by transfection and/or transduction. In some embodiments, a library construct is introduced into a host cell by viral-mediated transduction.

[00436] In some embodiments of the disclosed methods, provided host cells produce AAV vectors that are more functional and/or enhanced in an application, relative to a reference population. In some embodiments, provided host cells produce AAV vectors that are more functional and/or enhanced at transferring nucleic acid to a cell, relative to a reference population. In some embodiments, provided host cells produce AAV vectors that are more functional and/or enhanced therapeutically, relative to a reference population. In some embodiments, provided host cells produce AAV vectors that are more functional and/or enhanced in their intended application, relative to a reference population. In some embodiments, provided host cells comprise at least one synthetic sequence (e.g., encoding SHGP) that provides an increase in AAV vector production under a manufacturing practice relative to a reference cell population.

[00437] Disclosed herein is also a plurality of host cells permissive for AAV replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

[00438] (i) one or more AAV replication genes (SEQ ID NO: 1-4, 10, 12, 14);

[00439] (ii) one or more AAV capsid encoding genes (SEQ ID NO: 5-7, 11, 13, 15);

[00440] (iii) one or more viral helper genes (SEQ ID NO: 16-23); and

[00441] (iv) a nucleotide sequence operably linked to at least one functional AAV inverted terminal repeat (ITR) (SEQ ID NO: 8-9), wherein the nucleotide sequence encodes a payload (e.g., therapeutic gene);

[00442] wherein the Synthetic Helper Gene Product increases infectiousness (infectious unit titer) of AAV vectors produced by the plurality of host cells by at least 20%, 40%, 60%, 80%, 100%, 200%, or 500% compared to infectiousness of AAV vectors produced by a reference plurality of host cells that do not comprise the Synthetic Helper Gene Product.

[00443] In some embodiments, the presence of the Synthetic Helper Gene Product in the plurality of host cells is associated with an increase in AAV infectivity relative to a reference plurality of host cells that lacks the Synthetic Helper Gene Product. In some embodiments, the presence of the Synthetic Helper Gene Product in the plurality of host cells generates at least a 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, or 500% increase in AAV infectivity relative to a reference plurality of host cells that lacks the Synthetic Helper Gene Product.

[00444] In preferred embodiments, each host cell of the plurality of host cells is a mammalian host cell population.

[00445] In some embodiments, the payload is a therapeutic protein. In other embodiments, the payload is an RNA molecule.

[00446] In some embodiments, each host cell of the plurality of host cells produces the Synthetic Helper Gene Product.

[00447] In some embodiments, each host cell of the plurality of host cells comprises at least 10^A2 - 10^A6 AAV genomes per cell (10 - 10^A6 infectious particles per cell).

[00448] In some embodiments, each host cell of the plurality of host cells is configured to produce at least 10^A2 - 10^A6 AAV genomes per cell (10 - 10^A6 infectious particles per cell). [00449] In some embodiments, AAV nucleic acids of AAV vectors described herein typically include the cis-acting 5’ and 3’ ITR (SEQ ID NO: 8-9) sequences. In some embodiments, at least 80% of a typical ITR sequence (e.g., at least 85%, at least 90%, or at least 95%) is incorporated into constructs provided herein. Generally, ITRs are able to form a hairpin. The ability to form a hairpin can contribute to an ITR’s ability to self-prime, allowing primase-independent synthesis of a second DNA strand. ITRs can also aid in efficient encapsulation of an AAV construct in an AAV vector. An AAV ITR sequence may be obtained from any known AAV, including mammalian AAV types. In some embodiments, an ITR includes one or more modifications, e.g., truncations, deletions, substitutions or insertions, of a naturally occurring ITR sequence. In some embodiments, an ITR comprises fewer than 145 nucleotides.

[00450] In some embodiments, a barcode and/or a payload sequence is flanked by 5’ and 3’ AAV ITR (SEQ ID NO: 8-9) sequences. In some embodiments, an AAV nucleic acid comprises a barcode and a payload flanked by 5’ and 3’ AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types. [00451] In some embodiments, the plurality of host cells configured to produce an AAV vector composition of increased viral titer and/or transduction efficiency having TU: VG ratio from 1 : 100 to 1 :1. In some embodiments, TU: VG ratio of the AAV vector composition of increased viral titer and/or transduction efficiency is from 1 : 100 to 1 :50, from 1 : 50 to 1 :20, from 1 :20 to 1 : 10, from 1 : 10 to 1 :5, from 1 :5 to 1 :2, or from 1 :2 to 1 : 1.

[00452] In some embodiments, the Synthetic Helper Gene Product contains more than 10 amino acid residues, more than 100 residues, or more than 1000 residues.

[00453] In some embodiments, the nucleotide sequence positioned between two ITRs (SEQ ID NO: 8-9) comprises both a sequence that encodes a polypeptide that is a Synthetic Helper Gene Product during production in the host cell, and/or a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell. [00454] In some embodiments, the nucleotide sequence positioned between two ITRs (SEQ ID NO: 8-9) further encodes a reporter protein (e.g., GFP, luciferase) and/or a selectable marker (such as an antibiotic resistance gene, transcription factor, or other enzyme, which may allow to select for SHGP-mediated increase of payload gene expression) and/or a toxic marker. In further embodiments, the payload may be translationally fused to the Synthetic Helper Gene Product.

[00455] In some embodiments, the Synthetic Helper Gene Product is generated endogenously by the host cell.

[00456] Disclosed herein is also a method of producing a highly infectious adeno- associated virus (AAV) vector composition, the method comprising:

- I l l - [00457] (a) providing a plurality of host cells permissive for AAV replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

[00458] (i) one or more AAV replication genes (SEQ ID NO: 1-4, 10, 12, 14);

[00459] (ii) one or more AAV capsid encoding genes (SEQ ID NO: 5-7, 11, 13, 15);

[00460] (iii) one or more viral helper genes (SEQ ID NO: 16-23); and

[00461] (iv) a nucleotide sequence positioned between two inverted terminal repeats (ITRs) (SEQ ID NO: 8-9), wherein the nucleotide sequence encodes a payload (e.g. therapeutic gene);

[00462] wherein the Synthetic Helper Gene Product increases infectiousness (infectious unit titer) of AAV vectors produced by the plurality of host cells by at least 20%, 40%, 60%, 80%, 100%, 200%, or 500% compared to infectiousness (infectious unit titer) of AAV vectors produced by a reference plurality of host cells that do not comprise the Synthetic Helper Gene Product;

[00463] (b) incubating the plurality of host cells in a cell culture medium under suitable conditions, and collecting AAV vectors, thereby producing the AAV vector composition of increased viral titer and/or transduction efficiency, wherein the AAV vector composition of increased viral titer and/or transduction efficiency has an infectious unit titer which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than an infectious unit titer of a reference AAV vector composition produced without Synthetic Helper Gene Products.

[00464] In some embodiments, TU:VG ratio of the AAV vector composition of increased viral titer and/or transduction efficiency produced by the methods disclosed herein is from 1 : 100 to 1 :50, from 1 :50 to 1 :20, from 1 :20 to 1 : 10, from 1 : 10 to 1 :5, from 1 :5 to 1 :2, or from 1 :2 to 1 : 1.

[00465] In some embodiments, the AAV vector composition of increased viral titer and/or transduction efficiency has a viral genome titer which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral genome titer of a reference AAV vector composition produced without Synthetic Helper Gene Products.

[00466] In some embodiments, an AAV vector payload is less than 4 kb. In some embodiments, an AAV vector payload can include a sequence that is at least 500 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb. In some embodiments, an AAV vector payload can include a sequence that is at most 7.5 kb. In some embodiments, an AAV vector payload can include a sequence that is about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb. In some embodiments, an AAV vector can direct long- term expression of a payload. In other embodiments, an AAV vector can direct transient expression of a payload.

[00467] In some embodiments, the Synthetic Helper Gene Product is produced endogenously in the plurality of host cells and contains more than 10 amino acid residues.

[00468] In some embodiments, the one or more AAV replication genes (SEQ ID NO: 1- 4, 10, 12, 14) are those that encode proteins selected from the group consisting of: Rep78, Rep68, Rep52, and Rep40. In some embodiments, a combination of two or more AAV replication genes may be used.

[00469] In some embodiments, the one or more AAV capsid encoding genes (SEQ ID NO: 5-7, 11, 13, 15) are those that encode proteins selected from the group consisting of: VP1, VP2, and VP3. In some embodiments, a combination of two or more AAV capsid encoding genes may be used.

[00470] In some embodiments, the one or more viral helper genes (SEQ ID NO: 16-23) are selected from the group consisting of: Adenovirus El A, Adenovirus E1B55K, Adenovirus E2A, Adenovirus E4orf6, and Adenovirus VA. In some embodiments, a combination of two or more viral helper genes may be used.

[00471] In some embodiments, the first nucleotide sequence operably linked between two functional AAV inverted terminal repeats (ITRs) (SEQ ID NO: 8-9) comprises two or more sequences, wherein each of the two or more sequences encodes a unique Synthetic Helper Gene Product.

[00472] In some embodiments, the Synthetic Helper Gene Product does not impact efficiency of AAV production, but only impacts the stability of the host cell line that stably expresses the elements required for AAV production.

[00473] Mutagenesis of SHGPs (particularly at the CDRs or binding interface residues) can be used to create engineered molecules with specificity to new targets (e.g. protein targets or DNA sequences). Binding interactions of certain engineered proteins may perturb cellular behavior if expressed in living cells (e.g. preventing protein-protein interaction or altering gene expression). This binding protein-based perturbation strategy provides a novel means of manipulating cellular behavior.

[00474] A particularly useful and unexpected application of this perturbation strategy is the production of AAV and other viral vectors. It is unexpected that the co-expression of certain engineered binding proteins (e.g., mutagenized antibody-like proteins or transcription factors) in human cells which are simultaneously producing AAV particles, would lead to an improvement of AAV production. However, the inventors find unexpectedly that expression of SHGPs, such as antibody-like proteins (e.g. single domain antibodies, antibody mimetics), or transcriptional factors, provides substantial benefit enhancing characteristics of viral vector composition (see also Examples below).

[00475]

[00476] Viral genes essential for the replication of the virus in host cells

[00477] In some embodiments, the one or more AAV replication genes (SEQ ID NO: 1- 4, 10, 12, 14) are those that encode proteins selected from the group consisting of: Rep78, Rep68, Rep52, and Rep40

[00478] In some embodiments, the one or more AAV capsid encoding genes (SEQ ID NO: 5-7, 11, 13, 15) are those that encode proteins selected from the group consisting of: VP1, VP2, and VP3.

[00479] In some embodiments, the one or more adenovirus helper genes (SEQ ID NO: 16-23) are selected from the group consisting of: Adenovirus E1A, Adenovirus E1B55K, Adenovirus E2A, Adenovirus E4orf6, and Adenovirus VA.

[00480] In some embodiments, the one or more HSV helper genes (SEQ ID NO: 30-33) are selected from the group consisting of: UL5, UL8, UL52, ISHGP8. In other embodiments, the one or more HPV helper genes are selected from the group consisting of: El, E2, E6.

In yet other embodiments, the one or more HBoVl helper genes are selected from the group consisting of: NS2, NS4, NP1, BocaSR.

[00481] In some embodiments, each host cell of first, second, third or higher order plurality of host cells is a mammalian host cell. In other embodiments, each host cell of first, second, third or higher order plurality of host cells is an insect host cell.

[00482] In some embodiments, the one or more AAV replication genes are those that encode proteins selected from the group consisting of: Rep78, Rep68, Rep52, and Rep40 (SEQ ID NO: 1-4, 10, 12, 14).

[00483] In some embodiments, the one or more AAV capsid encoding genes are those that encode proteins selected from the group consisting of: VP1, VP2, and VP3 (SEQ ID NO: 5-7, 11, 13, 15).

[00484] In some embodiments, the one or more viral helper genes are selected from the group consisting of: Adenovirus El A, Adenovirus E1B55K, Adenovirus E2A, Adenovirus E4orf6, and Adenovirus VA (SEQ ID NO: 16-23).

[00485] In some embodiments, the viral helper genes critical for the replication, transcription, or packaging of a viral vector are derived from adenoviruses. These essential adenoviral helper genes, which may include but are not limited to Adenovirus El A, Adenovirus E1B55K, Adenovirus E2A, Adenovirus E4orf6, and Adenovirus VA, facilitate the processes essential for vector production. In particular embodiments, these helper genes can be supplied as part of the whole helper virus or as a subset of the genes.

[00486] In other embodiments, helper genes are obtained from herpesviruses (SEQ ID NO. 30-33). These herpes simplex virus (HSV) genes, including UL5, UL8, UL52, and the major DNA-binding protein UL29, have been found to provide helper gene function for AAV replication. In certain instances, the HSV-1 DNA polymerase complex, composed of UL30/UL42, is instrumental for AAV DNA replication. Other herpesvirus genera that support AAV replication, such as varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6), may be employed in specific embodiments.

[00487] In some embodiments, non-essential helper genes are sourced from Human Papillomavirus (HPV). The HPV El protein provides helper function analogous to AAV Rep78 but without Rep78’s endonuclease/covalent attachment activity. HPV genes El, E2, and E6 can be employed to boost rAAV and wt AAV replication and yield, particularly when used in combination with adenovirus helper genes.

[00488] In certain embodiments, non-essential helper genes are derived from Human Bocavirus (HBoV). Essential for AAV2 duplex DNA genome replication and progeny virion production are the HBoVl NP1 and NS4 proteins, and a newly identified viral long noncoding RNA, BocaSR. Specifically, HBoVl NS2, NP1, and BocaSR are required for productive infection of HEK293 and HeLa cells by AAV2.

[00489] In various embodiments, alternative helper genes are utilized from different viruses such as Herpes Simplex Virus (HSV) or Baculovirus to enhance vector production. For instance, in certain instances, HSV helper genes including UL5, UL8, ISHGP8, and ISHGP27 are employed. In other scenarios, Baculovirus helper genes like p80, pl43, p40, and p32 might be used. These alternative helper genes provide the flexibility to enhance replication and packaging processes.

[00490] Collectively, these embodiments reflect diverse sources of viral helper genes vital for the replication, transcription, or packaging of a viral vector, offering flexibility and adaptability in the production process.

[00491] In certain embodiments, derivatives of the Chinese Hamster Ovary (CHO) cell line are utilized. Known for their extensive application in biopharmaceutical production, these CHO cells provide a mammalian cell platform that can potentially be optimized for both AAV and lentiviral vector production. In some such embodiments, CHO cells are employed to produce advanced recombinant proteins, which necessitate proper protein folding and post- translational modifications. These CHO cell lines may further be engineered to enhance bioproduction efficiency and improve product quality.

[00492] In specific embodiments, the cell lines utilized for viral production, including adeno-associated virus (AAV) and lentivirus, originate from Human Embryonic Kidney 293 (HEK293) cells. These cells, along with their derivative or related strains, are routinely employed in biotechnology due to their notable transfection efficiency and ability to proliferate to high densities, thus facilitating efficient viral production. In particular embodiments, the HEK293 cell line is utilized as an expression host for proteins requiring human-specific post- translational modifications. Furthermore, these HEK293 cells may be used for the production of recombinant AAV and lentiviral particles, owing to the expression of the essential helper factors El A and E1B.

[00493] In some embodiments, the 293T cell line, an established lineage from the parental HEK293 line, may express the temperature sensitive allele of the large T antigen of Simian virus 40, contributing to improved recombinant protein production and therapeutic protein production. This cell line can potentially be used for both AAV and lentiviral production.

[00494] In some embodiments, the 293E cell line, another lineage derived from the parental HEK293 cell line, may express the Epstein-Barr virus nuclear antigen EBNA1, assisting in optimized recombinant protein production and the production of therapeutic proteins. These cells might also be suitable for AAV and lentivirus production.

[00495] In some embodiments, the 293 -F and 293 -H cell lines, industrially relevant suspension cell lines, are adapted for high-density suspension growth in serum-free medium, enabling large-scale cultivation and bioproduction in bioreactors with fast growth and high transfectivity. They can potentially be used for AAV and lentivirus production.

[00496] In some embodiments, the Freestyle 293-F cell line, a derivative of the HEK293 cell line, is adapted for high-density suspension growth in serum -free medium, facilitating large-scale production of therapeutic proteins in bioreactors due to its ability to increase volumetric cell density without cell clump formation. It can be employed for both AAV and lentivirus production.

[00497] In other embodiments, PER.C6 cells, a human retinal pigment epithelial cell line, are utilized for AAV and potentially lentivirus production. These cells, known for their easy maintenance and high transfection efficiency, offer a robust platform for the production of viral vectors. [00498] In certain embodiments, BTI-TN-5B1-4 cells, commonly known as High Five cells, are employed. These cells, derived from Trichoplusia ni, the cabbage looper, ovarian cells, can be used in the baculovirus expression vector system for AAV and potentially lentivirus production. Due to their capacity to grow to high densities in suspension culture and their reputation for high yield recombinant protein production, High Five cells serve as a powerful tool for viral production.

[00499] In some embodiments, Sf9 insect cells are used for viral production. As an established cell line for the baculovirus expression vector system, Sf9 cells offer a robust platform for recombinant protein production and AAV and potentially lentiviral vector generation.

[00500] In other embodiments, potential cell lines for viral production can include viro9 cells. While the utility of these cells for AAV and lentivirus production would require further investigation, their potential use contributes to the broad applicability of this approach.

[00501] In various embodiments, Baby Hamster Kidney (BHK) cells are employed for AAV and potentially lentivirus production. BHK cells can be transfected with plasmids encoding the viral vector genome, rep (SEQ ID NO: 1-4, 10, 12, 14) and cap genes (SEQ ID NO: 5-7, 11, 13, 15), and helper (SEQ ID NO: 16-23) functions from adenoviruses, making them particularly valuable for the production of certain viral serotypes.

[00502] Collectively, these embodiments reflect the diversity of cell lines that can be utilized for viral vector production, offering a range of options to accommodate specific requirements for AAV and lentivirus production.

[00503] Different AAV serotypes have different capsid protein sequences, replication protein sequences, ITR sequences, and other genes. These differences have large impacts on each serotype’s ability to infect different cell types; this is the primary motivation for use of different AAV serotypes. However, the same differences that provide enhanced targeting to one cell type over another also have large impacts on the manufacturability (e.g. viral genome titer, fulkempty ratio, infectivity /infectious titer, etc. . .). These benefits and challenges become even more pronounced when considering the use of chimeric, pseudotypes (e.g. AAV5 capsid with AAV2 genome elements), or machine-designed AAV vectors, which often do not fit neatly into any given serotype. This presents a problem for the clinical translational of AAV vectors to gene therapy products. For example, AAV serotypes AAV1-AAV9 produced in Hek293 cells or HeLa cells showed dramatically different infectious titers both within cell lines (e.g. across serotypes) and across cell lines (e.g. within serotypes). Considering the case of HEK293 cells (a preferred host cell line), AAV2 is produced at ~1E9 lU/ml (infectious units per ml), while AAV9 is produced at ~1E6 lU/ml - a 1000-fold difference.

[00504] When considering the productivity at the cell level, a nearly 3 order of magnitude range between different serotypes has been reported, spanning from -1000 viral genomes per cell (AAV4 produced in HEK293) to over -100,000 viral genomes per cell (AAV3 produced in Sf9 cells).

[00505] Thus, for one skilled in the art, it is readily apparent that there is substantial variation in the expected viral titer, infectious titer, and other properties of AAV products depending principally on the serotype (e.g. natural, chimeric, totally artificial), production process (e.g. cell line, triple transfection, helper virus), and potentially other aspects (e.g. the identity of the DNA payload to be deliver). The problem to be solved is to maximize viral yield (e.g. viral titer) and quality (e.g. infectiousness, fulkempty, etc. . .) for a variety of different therapeutic products that may have different starting points.

[00506] Thus, one skilled in the art will recognize that relative increases in viral titer, infectiousness, and other desirable properties are to be expected (e.g. because different AAV products will have dramatically different levels of manufacturing optimization required). One skilled in the art will reasonably conclude that a 2-fold increase in viral titer or infectiousness as compared to a reference viral composition, is both definite and provides substantial utility. One skilled in the art will further recognize that multiple 2-fold increases in viral titer or infectiousness (e.g. from a cell comprising multiple Synthetic Helper Gene Products), can exploit synergies that exist between different Synthetic Helper Gene Products.

[00507] In some embodiments, a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of a viral vector composition is disclosed herein; the Synthetic Helper Gene Product having between 30 and 300 amino acids and comprising at least one of the peptide motifs selected from the group consisting of the motifs provided in the motif table at the end of the specification in Example 14. In some embodiments, Synthetic Helper Gene Products can improve production of both lentivirus and AAV vector composition in the same host cell.

[00508] In some embodiments, the packaging system is a natural, replication-competent virus. In other embodiments, the packaging system is 2nd, 3rd, or 4^th lentiviral packaging system.

[00509] In some embodiments, Synthetic Helper Gene Product encoding sequences are part of a genetic circuit controlled by regulatory elements. [00510] In some embodiments, the Synthetic Helper Gene Product improves packaging of a toxic payload gene.

[00511] In some embodiments, a Synthetic Helper Gene Product encoding sequence is incorporated onto the lentivirus genome and integrated into the host cell genome.

[00512] In some embodiments, a payload of the lentivirus is a Synthetic Helper Gene Product encoding sequence that is operably linked to AAV ITRs (SEQ ID NO: 8-9).

[00513] In some embodiments, the Synthetic Helper Gene Products are discovered in a lentiviral enrichment scheme (e.g., Fig. 1 - Fig. 12 can be adapted to what is shown in Fig. 13). [00514] In some embodiments, the method is scaled up for large-scale industrial production of lentiviral vectors. This expansion might involve the use of industrial-scale bioreactors or other cell culture systems capable of handling large volumes, to enhance the production capacity of lentiviral vectors for widespread commercial or clinical use.

[00515] In some embodiments, the cells transduced by lentivirus packaged in the presence of Synthetic Helper Gene Product are human white blood cells.

[00516] In some embodiments, the lentivirus packaged in the presence of Synthetic Helper Gene Product harbors one or more of a Chimeric Antigen Receptor (CAR), T-Cell Receptor (TCR), cytokine, gene editing payload (e.g. cas9), interfering RNA, transcription factor.

[00517] In some embodiments, the technique can be employed for packaging replication-competent lentiviral vectors. This can enable the production of lentiviral vectors capable of multiple rounds of infection, which may be advantageous in certain research or therapeutic applications.

[00518] In some embodiments, the method can be used to enhance the production of lentiviral vectors for gene therapy applications. These can include treatments for genetic diseases, cancers, or other conditions that can benefit from gene-based therapies.

[00519] In some embodiments, the Synthetic Helper Gene Products can be designed to interact with specific viral or cellular proteins to boost packaging efficiency. This targeted interaction can enable a more efficient and effective packaging process.

[00520] In specific embodiments, the Synthetic Helper Gene Products can be incorporated into a packaging cell line to create a stable producer cell line for continuous production of lentiviral vectors. This can simplify the production process by eliminating the need for repeated transfections of the Synthetic Helper GeneS.

[00521] In some embodiments, the Synthetic Helper Gene Products can be utilized to enhance the production of lentiviral vectors for research applications, such as gene functional studies or disease modeling. This can significantly improve the capability and utility of lentiviral vectors as tools for biological research.

[00522] In some embodiments, the Synthetic Helper Genes are part of a lentiviral packaging kit. In some embodiments, the Synthetic Helper Gene Products are part of a lentiviral packaging kit.

[00523] In any of the embodiments herein, a kit disclosed herein may further comprise one or more additional components necessary for carrying out a method described herein, such as sample preparation reagents, buffers, labels, and the like. As such, the kits may include one or more containers such as vials or bottles, with each container containing one or more separate components of the kit, and reagents for carrying out one or more steps of a method described herein. The kits may also include a denaturation reagent, buffers such as binding buffers and hybridization buffers, wash mediums, enzyme substrates, reagents for generating a labeled molecule, negative and positive controls and written instructions for using the kit components for carrying out a method, for example, for analyzing a polypeptide as described herein. The instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub-packaging) etc. Any one or more of the kit components and instructions may be packaged, stored, and/or shipped separately from other kit components and instructions, or together with any one or more other kit components and instructions.

[00524] In some embodiments, pseudotyped lentiviral vectors, which use envelope proteins derived from other viruses to expand tropism, can be supplemented with Synthetic Helper Gene Products to further boost their transduction efficiency and target cell specificity. [00525] In some embodiments, in specific scenarios, chimeric lentiviral vectors, which combine elements from different viruses, may be augmented with Synthetic Helper Gene Products to enhance their specific characteristics, such as nuclear localization from Adeno- Associated Virus elements.

[00526] In various embodiments, self-inactivating (SIN) lentiviral vectors, known for their safety through modifications that render them self-inactivating, might utilize Synthetic Helper Gene Products to increase their packaging efficiency, maintaining their safety profile while improving lentiviral vector yield.

[00527] In some embodiments, inducible lentiviral vectors, which allow temporal control of transgene expression through inducible promoter elements, can benefit from the addition of Synthetic Helper Gene Products to further increase their overall efficiency and performance.

[0082] In some embodiments, the Synthetic Helper Gene Product enhances packaging of a chimeric or pseudotyped virus. In further embodiments, the chimeric or pseudotyped virus may be related.

[0083] In some embodiments, the Synthetic Helper Gene Product enhances packaging of a nested virus. In further embodiments the nested virus is an AAV virus nested inside of an Adenovirus. In further embodiments the nested virus is an AAV virus nested inside of an HSV. In further embodiments the nested virus is an AAV virus nested inside of an HPV. In further embodiments the nested virus is an AAV virus nested inside of a Lentivirus. In further embodiments the nested virus is an AAV virus nested inside of a Retrovirus. In further embodiments the nested virus is an AAV nested inside of a baculovirus. In further embodiments the nested virus is a lentivirus nested inside of an HSV. In further embodiments the nested virus is a retrovirus nested inside of an HSV. In further embodiments the nested virus is an Adenovirus nested inside of an HSV. In further embodiments the nested virus construct is able to support viral packaging of both viruses. In further embodiments the nested virus construct is integrated into the host cell genome. In further embodiments the nested virus construct is self-limiting. In further embodiments the nested virus construct is self-limiting through use of a chemical dependency. In further embodiments, the nested virus construct is self-limiting in certain cell lines, but not other cell lines. In further embodiments, the nested virus construct is self-limiting in certain cell lines, but not other cell lines based on the presence of Synthetic Helper Gene Products. In further embodiments, the nested virus construct is self-limiting in certain cell lines, but not other cell lines.

[0084] In some embodiments Synthetic Helper Gene Products that increase production of one virus serotype (e.g., AAV1) may also increase production of a second, related virus serotype (e.g., AAV2).

[0085] In some embodiments Synthetic Helper Gene Products that increase production of one virus (e.g., AAV) may also increase production of a second, unrelated virus (e.g., lentivirus).

[0086] In some embodiments, the Synthetic Helper Gene Product enrichment/discovery process may alternate between different viruses (e.g., AAV, Lentivirus, HSV, Adenovirus, Baculovirus) as well as different serotypes of different viruses (e.g., AAV1, AAV5, AAV8, AAV9, Adenovirus 1-5, HIV, FIV, BIV, HSV1, HSV2). In some embodiments, the Synthetic Helper Gene Product enrichment/discovery process may include enrichment for Synthetic Helper Gene Products that are indirectly related viral production. In some embodiments, Synthetic Helper Gene Products may be enriched for those that are non-toxic to host cells. [0087] In some embodiments, the Synthetic Helper Gene Product may enhance production of a provirus that is integrated onto the host cell genome. In further embodiments, the provirus is a lentivirus. In further embodiments, the provirus is a retrovirus. In further embodiments the provirus is an AAV. In further embodiments, the provirus is a nested virus. In further embodiments, the provirus is a non-integrating virus nested inside of a provirus.

[00528] Disclosed herein is also a viral packaging kit comprising at least one nucleotide sequence containing:

[00529] (i) at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

[00530] (ii) at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15);

[00531] (iii) at least one AAV helper gene (SEQ ID NO: 16-23); and

[00532] (iv) at least one gene that expresses a Synthetic Helper Gene Product, wherein

(i)-(iv) are provided on one or more expression plasmids and, when transfected into packaging cells, produces a viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector produced under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise (iv).

[00533] Disclosed herein is also a viral packaging kit comprising at least one nucleotide sequence containing:

[00534] (i) at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14);

[00535] (ii) at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15);

[00536] (iii) at least one AAV helper gene (SEQ ID NO: 16-23); and

[00537] (iv) at least one Synthetic Helper Gene Product reagent, wherein (i)-(iii) are provided on one or more expression plasmids and, when transfected into packaging cells with (iv), produces a viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector produced under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise (iv).

[00538] Disclosed herein is also a viral packaging cell line kit comprising the following: [00539] (i) a mammalian cell line;

[00540] (ii) at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14), genomically integrated; [00541] (iii) at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15), genomically integrated;

[00542] (iv) at least one AAV helper gene (SEQ ID NO: 16-23), genomically integrated; and

[00543] (v) at least one genomically integrated gene that expresses a Synthetic Helper

Gene Product, wherein, at least one of (i)-(v) is under inducible expression control and upon induction, the cell line produces a viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector produced under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the first plurality of host cells and does not comprise (v).

[00544] Disclosed herein is also a viral packaging kit comprising at least one nucleotide sequence containing:

[00545] (i) at least one lentiviral pol gene (SEQ ID NO: 25);

[00546] (ii) at least one lentiviral gag gene (SEQ ID NO: 26) and at least one env gene (SEQ ID NO: 27);

[00547] (iii) at least lentiviral rev gene (SEQ ID NO: 24); and

[00548] (iv) at least one gene that expresses a polypeptide, wherein (i)-(iv) are provided on one or more expression plasmids and, when transfected into packaging cells, produces a viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector produced under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise (iv).

[00549] Disclosed herein is also a viral packaging kit comprising at least one nucleotide sequence containing:

[00550] (i) at least one lentiviral pol gene (SEQ ID NO: 25);

[00551] (ii) at least one lentiviral gag gene (SEQ ID NO: 26) and at least one env gene (SEQ ID NO: 27);

[00552] (iii) at least lentiviral rev gene (SEQ ID NO: 24); and

[00553] (iv) at least one Synthetic Helper Gene Product reagent, wherein (i)-(iii) are provided on one or more expression plasmids and, when transfected into packaging cells with (iv), produces a viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector produced under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise (iv). [00554] Disclosed herein is also a viral packaging cell line kit comprising the following: [00555] (i) a mammalian cell line;

[00556] (ii) at least one AAV replication gene (SEQ ID NO: 1-4, 10, 12, 14), genomically integrated;

[00557] (iii) at least one AAV capsid encoding gene (SEQ ID NO: 5-7, 11, 13, 15), genomically integrated;

[00558] (iv) at least one AAV helper gene (SEQ ID NO: 16-23), genomically integrated; and

[00559] (v) at least one genomically integrated gene that expresses a Synthetic Helper

Gene Product, wherein, at least one of (i)-(v) is under inducible expression control and upon induction, the cell line produces a viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector produced under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the first plurality of host cells and does not comprise (v).

[00560] Disclosed herein is also a viral packaging cell line kit comprising the following: [00561] (i) a mammalian cell line;

[00562] (ii) at least one lentiviral pol gene (SEQ ID NO: 25), genomically integrated;

[00563] (iii) at least one lentiviral gag gene (SEQ ID NO: 26) and at least one env gene (SEQ ID NO: 27), genomically integrated;

[00564] (iv) at least one lentiviral rev gene (SEQ ID NO: 24), genomically integrated; and

[00565] (v) at least one genomically integrated gene that expresses a Synthetic Helper

Gene Product, wherein, at least one of (i)-(v) is under inducible expression control and upon induction, the cell line produces a viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector produced under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the first plurality of host cells and does not comprise (v).

[00566] Disclosed herein is also a kit that comprises at least one Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of a viral vector composition. In some embodiments, the kit disclosed herein is for obtaining a viral vector composition of increased viral titer and/or transduction efficiency. In some particular embodiments, the kit disclosed herein is for obtaining an AAV vector composition of increased viral titer and/or transduction efficiency. In other particular embodiments, the kit disclosed herein is for obtaining a lentivirus vector composition of increased viral titer and/or transduction efficiency. In some particular embodiments, the kit disclosed herein comprises at least one Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of a viral vector composition, the at least one Synthetic Helper Gene Product having between 30 and 300 amino acid residues and comprising one or more of the peptide motifs provided in the MOTIFS Table in Example 14.

[00567] In some embodiments, kits disclosed herein comprise at least one Synthetic Helper Gene Product and also comprise at least one non-peptide macromolecule, which is used together with the at least one Synthetic Helper Gene Product to deliver the Synthetic Helper Gene Product into the viral production cells in order to obtain a viral vector composition of increased viral titer and/or transduction efficiency. In some embodiments, the non-peptide macromolecule of the kit is selected from the group consisting of one or more cationic polymers, one or more cationic lipids, and one or more dendrimers. In some embodiments, the components of the kit disclosed herein are used to together in a transfection reagent to transfect a plurality of host cells to increase a characteristic of viral vectors produced by the plurality of host cells, wherein the characteristic of viral vectors is selected from the group consisting of: viral titer and transduction efficiency. In some embodiments, the non-peptide macromolecule of the kit comprises at least one of the moieties selected from the group consisting of: a linear or branched polyethyleneimine (PEI), PEI dendrimer, a polypropyleneimine (PPI), Poly(amidoamine) (PAA) and dendrimers (PAMAM), cationic cyclodextrin, polyalkylamine, a polyhydroxyalkylamine, poly(butyleneimine) (PBI), spermine, a N-substituted polyallylamine, N-substituted chitosan, a N-substituted polyornithine, a N-substituted polylysine (PLL), a N- substituted polyvinylamine, poly(P-amino ester), hyperbranched poly(amino ester) (h-PAE), networked poly(amino ester) (n-PAE), poly(4-hydroxy-l -proline ester) (PHP-ester), a poly-P- aminoacid, l,2-dioleoyl-3 -trimethylammonium -propane (DOTAP), l,2-dimyristoyl-3- trimethylammonium-propane (DMTAP), 3P-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l,2-dipalmitoyl-3 -trimethylammonium -propane (DPTAP), cationic liposome, and dendrimer. In some embodiments, the components of the kit (e.g., the Synthetic Helper Gene Product and at least one of the moieties listed above) are conjugated. In some embodiments, the components of the kit (e.g., the Synthetic Helper Gene Product and the at least one of the moieties listed above) are non-covalently associated with each other (e.g., through hydrogen bonding and/or electrostatic interactions, and so on). Non-limiting examples of dendrimers include poly(amidoamine) (PAMAM) dendrimers, polypropylene imine) (PPI) dendrimers, polyether-copolyester (PEPE) dendrimers, PEGylated dendrimers and peptide dendrimers. In some embodiments, the components of the kit are used to together in a transfection reagent which is used to deliver viral packaging plasmids into mammalian cells, and where the transfection results in at least a 2-fold higher yield of viral vector composition compared to a reference transfection reagent that that does not contain the SHGP.

[00568] In some embodiments of the disclosed methods, compositions or kits, the Synthetic Helper Gene Product comprises an amino acid sequence having at least 90%, at least 95% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 35 - SEQ ID NO: 221.

[00569] In some embodiments of the disclosed methods, compositions or kits, the Synthetic Helper Gene Product comprises an amino acid sequence selected from the group consisting of: HYD-HYD-HYD-POL-HYD, HYD-POL-HYD-HYD-HYD, HYD-HYD-HYD- HYD-HYD, HYD-HYD-POL-HYD-HYD, HYD-HYD-HYD-HYD-POL, POL-HYD-HYD- HYD-HYD, POL-HYD-HYD-HYD-POL, HYD-HYD-POS-HYD-HYD, HYD-HYD-HYD- POS-HYD, HYD-POL-POL-HYD-HYD, POL-HYD-HYD-POL-HYD, HYD-HYD-POL- HYD-POL, HYD-HYD-NEG-POL-HYD, HYD-HYD-NEG-HYD-HYD, HYD-NEG-POL- HYD-HYD, POS-POL-HYD-HYD-HYD, POL-HYD-POL-HYD-HYD, NEG-HYD-HYD- HYD-HYD, HYD-POL-POL-HYD-POL, and POL-HYD-POL-POL-HYD, wherein HYD is one of the following amino acid residues: 'G', 'A', 'V, T, 'L', 'M', 'P'; ARO is one of the following amino acid residues: 'F', 'W, 'Y'; POL is one of the following amino acid residues: 'S', 'T', 'Q', 'N', 'C; POS is one of the following amino acid residues: 'K', 'R', H'; and NEG is one of the following amino acid residues: 'D', 'E'.

[00570] The following enumerated embodiments are representative of the invention:

1. A method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of a viral vector composition, the method comprising: (a) culturing a first plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and (iv) a Synthetic Helper Gene Product encoded by a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the first nucleotide sequence within the viral capsids or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to the one or more viral-specific packaging sequences necessary for encapsulation of the second nucleotide sequence within the viral capsids, thereby obtaining a first plurality of viral vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps (bl)-(b2) one or more times in cycles:

(bl) allowing a plurality of viral vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for replication of the virus; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences producing the Synthetic Helper Gene Product, thereby obtaining a plurality of viral vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of viral vectors or the plurality of viral vectors of the present cycle to infect a final plurality of host cells; and

(d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of the viral vector composition by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from (i) the final plurality of host cells and/or (ii) a final plurality of viral vectors produced in the final plurality of host cells.

2. The method of embodiment 1, wherein in step (a), the first nucleotide sequence is operably linked to the one or more viral-specific packaging sequences, and the method comprises culturing the final plurality of host cells under conditions suitable for recombinant viral production, wherein each host cell of the final plurality of host cells comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to one or more viral-specific packaging sequences and producing the Synthetic Helper Gene Product encoded by the first nucleotide sequence, thereby producing the final plurality of viral vectors from the final plurality of host cells and determining the Synthetic Helper Gene Product by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from the final plurality of viral vectors.

3. The method of embodiment 1 or embodiment 2, wherein

(i) the viral vector composition is an adeno-associated virus (AAV) vector composition;

(ii) the at least one viral replication gene comprises at least one AAV replication gene (which encodes, for example, SEQ ID NO: 1-4, 10, 12, 14);

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene (which encodes, for example, SEQ ID NO: 5-7, 11, 13, 15);

(iv) the at least one additional viral gene comprises at least one AAV helper gene (which encodes, for example, SEQ ID NO: 16-23); and

(v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs) (for example, SEQ ID NO: 8-9).

4. The method of embodiment 1 or embodiment 2, wherein

(i) the viral vector composition is a lentivirus vector composition;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene (which encodes, for example, SEQ ID NO: 25);

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene (which encodes, for example, SEQ ID NO: 26) and at least one env gene (which encodes, for example, SEQ ID NO: 27);

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene (which encodes, for example, SEQ ID NO: 24); and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence (for example, SEQ ID NO: 34).

5. The method of any one of embodiments 1-4, wherein each host cell of first plurality of host cells and each host cell of final plurality of host cells are mammalian host cells.

6. The method of any one of embodiments 1-5, wherein the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences further encodes a reporter, a therapeutic payload or a selectable marker.

7. The method of any one of embodiments 1-6, wherein the first plurality of host cells at step (a) comprises at least 10,000 host cells each producing a unique Synthetic Helper Gene Product.

8. The method of any one of embodiments 1-7, further comprising (e): generating new viral vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the viral vector composition of increased viral titer and/or transduction efficiency.

9. The method of embodiment 8, which produces the viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from the group consisting of viral titer and transduction efficiency.

10. The method of embodiment 8 or embodiment 9, wherein the Synthetic Helper Gene Product is not essentially present in the viral vector composition of increased viral titer and/or transduction efficiency.

11. The method of any one of claims 8-10, wherein generating new viral vectors comprises: culturing a new plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the new plurality of host cells comprises the Synthetic Helper Gene Product determined in (d) and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload.

12. A plurality of host cells permissive for replication of a virus, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; wherein the Synthetic Helper Gene Product increases a characteristic of viral vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of viral vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of viral vectors is selected from the group consisting of viral titer and transduction efficiency.

13. The plurality of host cells of embodiment 12, wherein

(i) the virus is an adeno-associated virus (AAV);

(ii) the at least one viral replication gene comprises at least one AAV replication gene (which encodes, for example, SEQ ID NO: 1-4, 10, 12, 14);

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene (which encodes, for example, SEQ ID NO: 5-7, 11, 13, 15);

(iv) the at least one additional viral gene comprises at least one AAV helper gene (which encodes, for example, SEQ ID NO: 16-23); and

(v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs) (for example, SEQ ID NO: 8-9).

14. The plurality of host cells of embodiment 12, wherein

(i) the virus is a lentivirus;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene (which encodes, for example, SEQ ID NO: 25);

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene (which encodes, for example, SEQ ID NO: 26) and at least one env gene (which encodes, for example, SEQ ID NO: 27);

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene (which encodes, for example, SEQ ID NO: 24); and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence (for example, SEQ ID NO: 34).

15. The plurality of host cells of any one of embodiments 12-14, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

16. The plurality of host cells of any one of embodiments 12-15, wherein each host cell of the plurality of host cells is a mammalian host cell.

17. The plurality of host cells of any one of embodiments 12-16, wherein each host cell of the plurality of host cells is an insect host cell. 18. The plurality of host cells of any one of embodiments 12-17, wherein the payload comprises a therapeutic gene.

19. The plurality of host cells of any one of embodiments 12-18, wherein the plurality of host cells comprises at least 10,000 host cells.

20. A method of producing a viral vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(a) culturing a plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; and

(b) producing the viral vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the viral vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral titer and/or transduction efficiency of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

21. The method of embodiment 20, wherein

(i) the viral vector composition is an adeno-associated virus (AAV) vector composition;

(ii) the at least one viral replication gene comprises at least one AAV replication gene (which encodes, for example, SEQ ID NO: 1-4, 10, 12, 14);

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene (which encodes, for example, SEQ ID NO: 5-7, 11, 13, 15);

(iv) the at least one additional viral gene comprises at least one AAV helper gene (which encodes, for example, SEQ ID NO: 16-23); and

(v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs) (for example, SEQ ID NO: 8-9).

22. The method of embodiment 20, wherein (i) the viral vector composition is a lentivirus vector composition;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene (which encodes, for example, SEQ ID NO: 25);

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene (which encodes, for example, SEQ ID NO: 26) and at least one env gene (which encodes, for example, SEQ ID NO: 27);

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene (which encodes, for example, SEQ ID NO: 24); and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence (for example, SEQ ID NO: 34).

23. The method of any one of embodiments 20-22, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

24. The method of any one of embodiments 20-23, wherein the Synthetic Helper Gene Product is exogenously supplied to each host cell of the plurality of host cells.

25. The method of any one of embodiments 20-24, wherein each host cell of the plurality of host cells is a mammalian host cell.

26. The method of any one of embodiments 20-25, wherein each host cell of the plurality of host cells is an insect host cell.

27. The method of any one of embodiments 20-26, wherein the plurality of host cells comprises at least 10,000 host cells.

28. The method of any one of embodiments 20-27, wherein the Synthetic Helper Gene Product is not essentially present in the viral vector composition of increased viral titer and/or transduction efficiency.

29. A method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of adeno-associated virus (AAV) vector composition, the method comprising:

(a) culturing a first plurality of host cells permissive for AAV replication under conditions suitable for recombinant AAV production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one AAV replication gene;

(ii) at least one AAV capsid encoding gene;

(iii) at least one AAV helper gene; and

(iv) a Synthetic Helper Gene Product produced from a first nucleotide sequence, wherein

(v) the first nucleotide sequence is operably linked to at least two functional AAV inverted terminal repeats (ITRs) (for example, SEQ ID NO: 8-9) or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to at least two functional AAV ITRs (for example, SEQ ID NO: 8-9), thereby generating a first plurality of AAV vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of AAV vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for AAV replication; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant AAV production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the at least two functional AAV ITRs producing the Synthetic Helper Gene Product, thereby generating a plurality of AAV vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of AAV vectors or the plurality of AAV vectors of the present cycle to infect a final plurality of host cells; and

(d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of AAV vector composition by analyzing nucleotide sequences operably linked to the at least two functional AAV inverted terminal repeats (ITRs) from (i) the final plurality of host cells and/or (ii) a final plurality of AAV vectors produced in the final plurality of host cells.

30. The method of embodiment 29, wherein in step (a), the first nucleotide sequence is operably linked to the at least two functional AAV ITRs, and the method comprises culturing the final plurality of host cells under conditions suitable for recombinant AAV production, wherein each host cell of the final plurality of host cells comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the at least two functional AAV ITRs and producing the Synthetic Helper Gene Product encoded by the first nucleotide sequence, thereby producing the final plurality of AAV vectors from the final plurality of host cells and obtaining the Synthetic Helper Gene Product by analyzing nucleotide sequences operably linked to the at least two functional AAV ITRs from the final plurality of AAV vectors. 31. The method of embodiment 29 or embodiment 30, wherein the Synthetic Helper Gene Product produced during AAV production of the polypeptide contains more than 6 and less than 300 amino acid residues, excluding an essentially synthetic solubility domain.

32. The method of any one of embodiments 29-31, wherein each host cell of first plurality of host cells and each host cell of final plurality of host cells are mammalian host cells.

33. The method of any one of embodiments 29-32, wherein the first nucleotide sequence operably linked to the at least two functional AAV ITRs further encodes a reporter, a therapeutic payload or a selectable marker.

34. The method of any one of embodiments 29-33, wherein the first plurality of host cells at step (a) comprises at least 10,000 host cells each producing a unique Synthetic Helper Gene Product.

35. The method of any one of embodiments 29-34, further comprising (e): generating new AAV vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the AAV vector composition of increased viral titer and/or transduction efficiency.

36. The method of embodiment 35, which produces the AAV vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference AAV vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from the group consisting of viral titer and transduction efficiency.

37. The method of embodiment 35, wherein the Synthetic Helper Gene Product is not essentially present in the AAV vector composition of increased viral titer and/or transduction efficiency.

38. The method of any one of embodiments 29-37, wherein the Synthetic Helper Gene Product comprises an amino acid sequence having at least 90%, at least 95% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 35 - SEQ ID NO: 221.

39. A plurality of host cells permissive for AAV replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises: (i) at least one AAV replication gene (which encodes, for example, SEQ ID NO: 1-4, 10, 12, 14); (ii) at least one AAV capsid encoding gene (which encodes, for example, SEQ ID NO: 5-7, 11, 13, 15);

(iii) at least one AAV helper gene (which encodes, for example, SEQ ID NO: 16-23); and

(iv) a nucleotide sequence operably linked to at least two functional AAV internal terminal repeats (ITRs) (for example, SEQ ID NO: 8-9), wherein the nucleotide sequence encodes a payload; wherein the Synthetic Helper Gene Product increases a characteristic of AAV vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of AAV vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of AAV vectors is selected from the group consisting of viral titer and transduction efficiency.

40. The plurality of host cells of embodiment 39, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

41. The plurality of host cells of embodiment 39 or embodiment 40, wherein the Synthetic Helper Gene Product is exogenously supplied to each host cell of the plurality of host cells.

42. The plurality of host cells of any one of embodiments 39-41, wherein the Synthetic Helper Gene Product contains more than 6 and less than 300 amino acid residues.

43. The plurality of host cells of any one of embodiments 39-42, wherein each host cell of the plurality of host cells is a mammalian host cell.

44. The plurality of host cells of any one of embodiments 39-43, wherein each host cell of the plurality of host cells is an insect host cell.

45. The plurality of host cells of any one of embodiments 39-44, wherein the payload comprises a therapeutic gene.

46. The plurality of host cells of any one of embodiments 39-45, wherein the plurality of host cells comprises at least 10,000 host cells.

47. The plurality of host cells of any one of embodiments 39-46, wherein the Synthetic Helper Gene Product comprises an amino acid sequence having at least 90%, at least 95% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 35 - SEQ ID NO: 221.

48. A method of producing an adeno-associated virus (AAV) vector composition of increased viral titer and/or transduction efficiency, the method comprising: (a) culturing a plurality of host cells permissive for AAV replication under conditions suitable for recombinant AAV production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one AAV replication gene (which encodes, for example, SEQ ID NO: 1-4, 10, 12, 14);

(ii) at least one AAV capsid encoding gene (which encodes, for example, SEQ ID NO: 5-7, 11, 13, 15);

(iii) at least one AAV helper gene (which encodes, for example, SEQ ID NO: 16-23); and

(iv) a nucleotide sequence operably linked between two functional AAV internal terminal repeats (ITRs) (for example, SEQ ID NO: 8-9), wherein the nucleotide sequence encodes a payload; and

(b) producing the AAV vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the AAV vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than an viral titer and/or transduction efficiency of a reference AAV vector composition produced from a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

49. The method of embodiment 48, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

50. The method of embodiment 48, wherein the Synthetic Helper Gene Product is exogenously supplied to each host cell of the plurality of host cells.

51. The method of any one of embodiments 48-50, wherein each host cell of the plurality of host cells is a mammalian host cell.

52. The method of any one of embodiments 48-51, wherein each host cell of the plurality of host cells is an insect host cell.

53. The method of any one of embodiments 48-52, wherein the plurality of host cells comprises at least 10,000 host cells.

54. The method of any one of embodiments 48-53, wherein the Synthetic Helper Gene Product is not essentially present in the AAV vector composition of increased viral titer and/or transduction efficiency.

55. The method of any one of embodiments 48-54, wherein the Synthetic Helper Gene Product comprises an amino acid sequence having at least 90%, at least 95% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 35 - SEQ ID NO: 221.

56. A method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of lentivirus vector composition, the method comprising:

(a) culturing a first plurality of host cells permissive for lentivirus replication under conditions suitable for recombinant lentivirus production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one lentiviral gag gene (which encodes, for example, SEQ ID NO: 26);

(ii) at least one lentiviral pol gene (which encodes, for example, SEQ ID NO: 25);

(iii) at least one lentiviral rev gene (which encodes, for example, SEQ ID NO: 24);

(iv) at least one env gene (which encodes, for example, SEQ ID NO: 27); and

(v) a Synthetic Helper Gene Product produced from a first nucleotide sequence, wherein

(vi) the first nucleotide sequence is operably linked to a Psi sequence (for example, SEQ ID NO: 34) or (vii) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to a Psi sequence (for example, SEQ ID NO: 34), thereby obtaining a first plurality of lentivirus vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps one or more times in cycles:

(bl) allowing a plurality of lentivirus vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for lentivirus replication; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant lentivirus production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iv) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the Psi sequence producing the Synthetic Helper Gene Product, thereby obtaining a plurality of lentivirus vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of lentivirus vectors or the plurality of lentivirus vectors of the present cycle to infect a final plurality of host cells; and

(d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of lentivirus vector composition by analyzing nucleotide sequences operably linked to the Psi sequence from (i) the final plurality of host cells and/or (ii) a final plurality of lentivirus vectors produced in the final plurality of host cells. 57. The method of embodiment 56, wherein in step (a), the first nucleotide sequence is operably linked to the Psi sequence, and the method comprises culturing the final plurality of host cells under conditions suitable for recombinant lentivirus production, wherein each host cell of the final plurality of host cells comprises the elements (i)-(iv) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the Psi sequence and producing the Synthetic Helper Gene Product encoded by the first nucleotide sequence, thereby producing the final plurality of lentivirus vectors from the final plurality of host cells and determining the Synthetic Helper Gene Product by analyzing nucleotide sequences operably linked to the Psi sequence from the final plurality of lentivirus vectors.

58. The method of embodiment 57 or embodiment 56, wherein each host cell of first plurality of host cells and each host cell of final plurality of host cells are mammalian host cells.

59. The method of any one of embodiments 56-58, wherein the first nucleotide sequence operably linked to the Psi sequence further encodes a reporter, a therapeutic payload or a selectable marker.

60. The method of any one of embodiments 56-59, wherein the first plurality of host cells at step (a) comprises at least 10,000 host cells each producing a unique Synthetic Helper Gene Product.

61. The method of any one of embodiments 56-60, further comprising (e): generating new lentivirus vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the lentivirus vector composition of increased viral titer and/or transduction efficiency.

62. The method of embodiment 61, which produces the lentivirus vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference lentivirus vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from the group consisting of viral titer and transduction efficiency.

63. The method of embodiment 61, wherein the Synthetic Helper Gene Product is not essentially present in the lentivirus vector composition of increased viral titer and/or transduction efficiency. 64. A plurality of host cells permissive for lentivirus replication, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one lentiviral gag gene (which encodes, for example, SEQ ID NO: 26);

(ii) at least one lentiviral pol gene (which encodes, for example, SEQ ID NO: 25);

(iii) at least one lentiviral rev gene (which encodes, for example, SEQ ID NO: 24)

(iv) at least one env gene (which encodes, for example, SEQ ID NO: 27); and

(v) a nucleotide sequence operably linked to a Psi sequence (for example, SEQ ID NO: 34), wherein the nucleotide sequence encodes a payload (e.g., therapeutic gene); wherein the Synthetic Helper Gene Product increases a characteristic of lentivirus vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of lentivirus vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(v) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of lentivirus vectors is selected from the group consisting of viral titer and transduction efficiency.

65. The plurality of host cells of embodiment 64, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

66. The plurality of host cells of embodiment 64, wherein the Synthetic Helper Gene Product is exogenously supplied to each host cell of the plurality of host cells.

67. The plurality of host cells of any one of embodiments 64-66, wherein each host cell of the plurality of host cells is a mammalian host cell.

68. The plurality of host cells of any one of embodiments 64-67, wherein the plurality of host cells comprises at least 10,000 host cells.

69. The plurality of host cells of any one of embodiments 64-68, wherein the payload comprises a therapeutic gene.

70. A method of producing a lentivirus vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(a) culturing a plurality of host cells permissive for lentiviral replication under conditions suitable for recombinant lentiviral production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one lentiviral gag gene (which encodes, for example, SEQ ID NO: 26);

(ii) at least one lentiviral pol gene (which encodes, for example, SEQ ID NO: 25);

(iii) at least one lentiviral rev gene (which encodes, for example, SEQ ID NO: 24); (iv) at least one env gene (which encodes, for example, SEQ ID NO: 27); and

(v) a nucleotide sequence operably linked to a Psi sequence (for example, SEQ ID NO: 34), wherein the nucleotide sequence encodes a payload (e.g., therapeutic gene); and

(b) producing the lentivirus vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the lentivirus vector composition has an increased viral titer and/or transduction efficiency which is at least a 20%, 40%, 60%, 80%, 100%, 200%, or 500% greater than a viral titer and/or transduction efficiency of a reference lentivirus vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(v) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

71. The method of embodiment 70, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

72. The method of embodiment 70, wherein the Synthetic Helper Gene Product is exogenously supplied to each host cell of the plurality of host cells.

73. The method of any one of embodiments 70-72, wherein each host cell of the plurality of host cells is a mammalian host cell.

74. The method of any one of embodiments 70-73, wherein each host cell of the plurality of host cells is an insect host cell.

75. The method of any one of embodiments 70-74, wherein the plurality of host cells comprises at least 10,000 host cells.

76. The method of any one of embodiments 70-75, wherein the Synthetic Helper Gene Product is not essentially present in the lentivirus vector composition of increased viral titer and/or transduction efficiency.

77. A kit for obtaining a viral vector composition of increased viral titer and/or transduction efficiency, comprising at least one Synthetic Helper Gene Product and a nonpeptide macromolecule selected from the group consisting of: a linear or branched polyethyleneimine (PEI), PEI dendrimer, a polypropyleneimine (PPI), Poly(amidoamine) (PAA) and dendrimers (PAMAM), cationic cyclodextrin, polyalkylamine, a polyhydroxyalkylamine, poly(butyleneimine) (PBI), spermine, a N-substituted polyallylamine, N-substituted chitosan, a N-substituted polyomithine, a N- substituted polylysine (PLL), a N-substituted polyvinylamine, poly(P-amino ester), hyperbranched poly(amino ester) (h-PAE), networked poly(amino ester) (n-PAE), poly(4- hydroxy-1 -proline ester) (PHP-ester), a poly-P-aminoacid, l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 3P-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), N-[l- (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dipalmitoyl- 3 -trimethylammonium-propane (DPTAP), cationic liposome, and dendrimer.

78. A plurality of host cells permissive for replication of a virus, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; wherein the plurality of host cells comprises at least 100,000, at least 500,000, at least 1,000,000, or at least 10,000,000 host cells each comprising structurally different Synthetic Helper Gene Products.

79. The plurality of host cells of embodiment 78, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

80. The plurality of host cells of embodiment 79, wherein at least one hundred of structurally different Synthetic Helper Gene Products produced in the at least 1,000,000 host cells each increases a characteristic of viral vectors produced by the host cell by at least 2-fold compared to a corresponding characteristic of viral vectors produced by a reference host cell under essentially identical conditions, wherein the reference host cell comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of viral vectors is selected from the group consisting of viral titer and transduction efficiency.

81. The plurality of host cells of embodiment 78 or embodiment 79, wherein

(i) the virus is an adeno-associated virus (AAV);

(ii) the at least one viral replication gene comprises at least one AAV replication gene (which encodes, for example, SEQ ID NO: 1-4, 10, 12, 14);

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene (which encodes, for example, SEQ ID NO: 5-7, 11, 13, 15);

(iv) the at least one additional viral gene comprises at least one AAV helper gene (which encodes, for example, SEQ ID NO: 16-23); and (v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs) (for example, SEQ ID NO: 8-9).

82. The plurality of host cells of embodiment 78 or embodiment 79, wherein

(i) the virus is a lentivirus;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene (which encodes, for example, SEQ ID NO: 25);

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene (which encodes, for example, SEQ ID NO: 26) and at least one env gene (which encodes, for example, SEQ ID NO: 27);

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene (which encodes, for example, SEQ ID NO: 24); and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence (for example, SEQ ID NO: 34).

83. The plurality of host cells of embodiment 78, wherein each host cell of plurality of host cells are mammalian host cells.

84. The method, plurality of host cells or kit of any one of embodiments 1-83, wherein the Synthetic Helper Gene Product is not present, or is not essentially present, in the viral vector composition of increased viral titer and/or transduction efficiency.

85. The method, plurality of host cells or kit of any one of embodiments 1-84, wherein the Synthetic Helper Gene Product comprises between 6 and 300 amino acid residues, and further comprises one or more of the peptide motifs listed in the Synthetic Helper Motifs table provided in Example 14.

86. The method, plurality of host cells or kit of any one of embodiments 1-85, wherein the Synthetic Helper Gene Product comprises an affinity purification tag.

87. The method, plurality of host cells or kit of any one of embodiments 1-87, wherein the affinity purification tag is configured to separate (e.g., via affinity purification) the Synthetic Helper Gene Product from other components of a host cell that produces the Synthetic Helper Gene Product.

88. The method, plurality of host cells or kit of any one of embodiments 1-87, wherein the Synthetic Helper Gene Product comprises a nanobody.

89. The method, plurality of host cells or kit of any one of embodiments 1-87, wherein the Synthetic Helper Gene Product comprises an amino acid sequence selected from the group consisting of: HYD-HYD-HYD-POL-HYD, HYD-POL-HYD-HYD-HYD, HYD-HYD- HYD-HYD-HYD, HYD-HYD-POL-HYD-HYD, HYD-HYD-HYD-HYD-POL, POL- HYD-HYD-HYD-HYD, POL-HYD-HYD-HYD-POL, HYD-HYD-POS-HYD-HYD, HYD-HYD-HYD-POS-HYD, HYD-POL-POL-HYD-HYD, POL-HYD-HYD-POL-HYD, HYD-HYD-POL-HYD-POL, HYD-HYD-NEG-POL-HYD, HYD-HYD-NEG-HYD-HYD, HYD-NEG-POL-HYD-HYD, POS-POL-HYD-HYD-HYD, POL-HYD-POL-HYD-HYD, NEG-HYD-HYD-HYD-HYD, HYD-POL-POL-HYD-POL, and POL-HYD-POL-POL- HYD, wherein HYD is one of the following amino acid residues: 'G', 'A', 'V, T, 'L', 'M', 'P'; ARO is one of the following amino acid residues: 'F', 'W, 'Y'; POL is one of the following amino acid residues: 'S', 'T', 'Q', 'N', 'C; POS is one of the following amino acid residues: 'K', 'R', 'H'; and NEG is one of the following amino acid residues: 'D', E'.

90. A method of producing a viral vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(A) obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of a viral vector composition by performing the following steps:

(a) culturing a first plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a Synthetic Helper Gene Product encoded by a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the first nucleotide sequence within the viral capsids or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to the one or more viral-specific packaging sequences necessary for encapsulation of the second nucleotide sequence within the viral capsids, thereby obtaining a first plurality of viral vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps (bl)-(b2) one or more times in cycles:

(bl) allowing a plurality of viral vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for replication of the virus; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences producing the Synthetic Helper Gene Product, thereby obtaining a plurality of viral vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of viral vectors or the plurality of viral vectors of the present cycle to infect a final plurality of host cells; and

(d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of the viral vector composition by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from (i) the final plurality of host cells and/or (ii) a final plurality of viral vectors produced in the final plurality of host cells; and

(B) generating new viral vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the viral vector composition of increased viral titer and/or transduction efficiency.

91. The method of embodiment 90, which produces the viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from the group consisting of viral titer and transduction efficiency.

92. The method of embodiment 90, wherein the Synthetic Helper Gene Product is not essentially present in the viral vector composition of increased viral titer and/or transduction efficiency.

93. The method of embodiment 90, wherein the first plurality of host cells comprises at least 100,000 host cells each comprising structurally different Synthetic Helper Gene Products.

94. The method of embodiment 90, wherein

(i) the viral vector composition is an adeno-associated virus (AAV) vector composition;

(ii) the at least one viral replication gene comprises at least one AAV replication gene;

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene;

(iv) the at least one additional viral gene comprises at least one AAV helper gene; and (v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs).

95. The method of embodiment 90, wherein

(i) the viral vector composition is a lentivirus vector composition;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene;

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene and at least one env gene;

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene; and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence.

96. The method of embodiment 90, wherein each host cell of first plurality of host cells and each host cell of final plurality of host cells are mammalian host cells.

97. The method of embodiment 90, wherein the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences further encodes a reporter, a therapeutic payload or a selectable marker.

98. The method of embodiment 90, wherein generating new viral vectors comprises: culturing a new plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the new plurality of host cells comprises the Synthetic Helper Gene Product determined in (d) and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload.

[00571] EXAMPLES

[00572] Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way. Certain aspects of the present invention, including, but not limited to, general cell culture propagation methods, general methods and reagents of producing recombinant AAV vectors in host cells, AAV vector extraction from host cells, generating and screening libraries of protein mutants were disclosed in the earlier publications: Yeast surface display platform for rapid discovery of conformationally selective nanobodies, 2021; Short peptides act as inducers, anti-inducers and corepressors of Tet repressor, 2012; A Novel TetR- Regulating Peptide Turns off rtTA-Mediated Activation of Gene Expression, 2014; Construction of Peptide Library in Mammalian Cells by dsDNA-Based Strategy, 2023; Construction of a linker library with widely controllable flexibility for fusion protein design, 2015; Rapid construction of metabolite biosensors using domain-insertion profiling, 2016; Genome-wide activation screens to increase adeno-associated virus production, 2021. [00573] Example 1. Improving AAV vectors manufacturability by Synthetic Helper Gene Products discovered through a high throughput screen of mutational library.

[00574] Host cell lines used for manufacturing of AAV vectors are not optimized for AAV production. The cellular environment can be efficiently modulated by intracellular Synthetic Helper Gene Products, which can affect AAV packaging and infectivity in a variety of ways. Endogenously generated Synthetic Helper Gene Products are advantageous in that they do not need to cross the cell membrane because they are expressed intracellularly. This eliminates the usual restrictions related to small molecule discovery that requires compounds to be able to cross the cell membrane in order to modulate intracellular targets. Synthetic Helper Gene Products have a variety of advantages over small molecules. Firstly, their modularity makes their construction simple. Second, they are genetically encoded and ribosomally expressed, ensuring high local concentration. Third, because the Synthetic Helper Gene Product diversity scales exponentially, diversity is practically limitless. Fourth, because Synthetic Helper Gene Products can be encoded on DNA and generated ribosomally it is easy to link biochemical function to chemical identity. Fifth, because Synthetic Helper Gene Products can be encoded on DNA and generated ribosomally, these mutant libraries can be “selected” as opposed to screened (e.g. traditional drug discovery). Sixth, Synthetic Helper Gene Products can be tagged with purification tags, making their removal easier compared to small molecules.

[00575] Because mechanisms by which Synthetic Helper Gene Products may increase AAV packaging and infectivity during viral particle assembly/packaging in the host cell are diverse (e.g. increasing/decreasing favorability of capsid stoichiometries, altering post translational modification, altering rAAV DNA payload methylation, altering DNA packaging, altering full: empty ratios, altering proteolytic process, altering intracellular trafficking, altering exposure history to different pH levels, altering allosteric signaling networks of capsid proteins/assemblies, altering rAAV viral escape, altering transcriptional profiles of specific genes or globally, inhibiting genes that interfere with viral synthesis, activating genes that enhance viral synthesis, and others) and have not been fully explored, a high throughput, competitive enrichment of an rAAV-vectored Synthetic Helper Gene Product library was performed to select for Synthetic Helper Gene Products that enhanced the yield and/or functional properties of rAAV.

[00576] SHGP Enrichment Overview: To perform a selective enrichment of AAV manufactuability enhancing Synthetic Helper Gene Products in host cells permissive for AAV replication, a genetically encoded Synthetic Helper Gene Product library was inserted into the AAV packaging cell line (e.g., a cell containing the components necessary for AAV biosynthesis) flanked by AAV ITRs (SEQ ID NO: 8-9); and the SHGPs were transcribed and translated within the cell. The presence of a unique Synthetic Helper Gene Product in each host cell resulted in different types and different degrees of modulation to various cellular components (e.g. inactivating a hydrolase, hyper stabilizing a cofactor regeneration enzyme, altering transcription factor activity, altering stress response, etc.). DNAs encoding/identifying specific Synthetic Helper Gene Products expressed in the host cell were packaged in AAV capsids, where each capsid contained DNA encoding/identifying the specific Synthetic Helper Gene Product. The host cell’s ability to efficiently produce highly functional AAV vectors comprising DNA that encodes/identifies Synthetic Helper Gene Products is influenced by the cell’s physiological state, which can be modulated by the Synthetic Helper Gene Product. The cell’s molecular genetic state may become more or less favorable to rAAV viral synthesis as a result of the presence of the Synthetic Helper Gene Product. The cells then produced AAV capsids harboring SHGs; SHGs that enhanced viral packaging gain a selective advantage and become over represented in the first round of enrichment (Fig. 1 Parts A-D; Fig. 2 Parts A-C; Fig. 4 Parts A-D; Fig. 5; Fig. 9; Fig. 11 Part A; Fig. 16; Part B of Figs. 25, 27, and 29-30.). A second round of selection enables identification of SHGs with other useful properties. The AAV capsids that were produced in the first round of enrichment were used to infect naive host cells permissive for AAV replication for a second round of SHG selection. Infection by these AAVs appeared to proceed with varying efficiency, where the efficiency of the infection was determined by infectivity -modulating properties of the specific peptides during the first round of packaging. Peptides that positively modulated AAV packaging and infectivity resulted in increased infection rates for corresponding AAV vectors, in which DNAs encoding these peptides were present (either through enhanced yield or enhanced transduction efficiency). Thus, proportions of host cells having AAV manufacturability-enhancing Synthetic Helper Gene Product gene sequences were increased after one round of infection (see Fig. 16, Fig. 26 Parts A and B, and Fig. 28 Parts A and B for round 2 selections of AAV-vectored SHGP libraries and enrichment). Similarly, proportions of host cells having AAV-manufacturability- reducing Synthetic Helper Gene Product gene sequences were depleted after one round of infection (see Fig. 11 for conceptual description, points less than 0 on the X axis in Fig. 16, and points less than 0 on the X axis in Figs. 25-33 Part B). Proportions of host cells having Synthetic Helper Gene Product gene sequences with little or no effect on rAAV manufacturability were reduced after one round of infection because Synthetic Helper Gene Product gene sequences that enhanced rAAV manufacturability were able to out-compete neutral Synthetic Helper Gene Products (see enrichment histograms Figs. 25-33 Part B). After the first infection round, resulting AAV particles were collected and used for infection of new naive host cells permissive for AAV replication (the second infection round). Again, during the second infection round, AAV viral particles of higher infectivity levels (due to effect of the peptide on the packaging cell), and/or AAV viral particles that more efficiently packaged the Synthetic Helper Gene DNA sequence, were able to infect more naive cells and/or more effectively deliver/transduce the DNA encoding/identifying the peptide; thus, populations of host cells having AAV-enhancing peptides were further enriched (The vertical bulges in the histograms of Figs. 26 and 28 Part A illustrate this clearly).

[00577] In contrast, Synthetic Helper Gene sequences that negatively affected AAV manufacturability were packaged less efficiently and/or were packaged in less infectious, lower quality capsids. These capsids were less capable of infecting naive cells. The DNA encoding these less efficacious Synthetic Helper Gene Products were produced, packaged, and/or transduced less effectively and were effectively eliminated from host cell culture after 2-3 reinfections (e.g., outcompeted).

[00578] The described process has been repeated 2-4 times using up to 7 different Synthetic Helper Gene Product library architectures (2 antibody-like architectures and 5 engineered transcription factor variants), and different enrichment strategies or conditions (e.g. media, transfection reagent, cell lines, etc. . .) as is typical for those skilled in the art who are performing process development. rAAVs harboring DNA encoding Synthetic Helper Gene Products that increased AAV packaging and infectivity were successfully selected from a starting library of mutagenized Synthetic Helper Gene Products. Thus, genetically encoded, virally vectored Synthetic Helper Gene libraries can be used to select for AAV performance enhancing Synthetic Helper Gene Products. Technical details of the described screen are provided below.

[00579] We performed this process on a variety of different types of exemplary Synthetic Helper Gene Products, Nanobody CDR3 libraries (exemplary antibody -like SHGPs) and error prone PCR mutagenized engineered transcription factors. Diagrams of the designs are provided in Figs. 14 and 15. Preferred enrichment and characterization workflows are visualized in Figs. 1-12. Exemplary results are provided in Figs. 16-34.

[00580] More specifically, several classes of SHGP libraries were created, spanning antibody-like protein SHGPs and engineered transcription factor SHGPs. For antibody-like SHGPs, architecture details are provided in Fig. 14 and, briefly, libraries were constructed via saturation mutagenesis, targeting CDR3. As exemplary antibody-like SHGPs, two nanobody scaffolds were mutagenized (SEQ ID NOs: 215-216). For engineered transcription factor SHGPs, architecture details are provided in Fig. 15 and, briefly, the parental transcription factors (SEQ ID NOs: 217-221) containing a NLS sequence were subjected to random mutagenesis via error prone PCR.

[00581] These sequences were inserted into a standard AAV transfer plasmid between two AAV ITRs (SEQ ID NO: 8-9) along with an eGFP for visualization. HEK-293T cells (50- 70% confluent, ~10^A6 cells) grown in DMEM with 5% FBS were triple transfected with pHelper Vector, AAV transfer plasmid encoding SHGP library, and AAVpro® Packaging Rep-Cap Plasmid (AAV2) using JetOptimus Transfection Reagent. After 24 hours, the medium was changed to DMEM with 2% FBS, and on day 3 post-transfection, eGFP expression was quantified, and cells were lysed with a citric acid buffer for AAV extraction and purification. Subsequent enrichment involved transducing 50-70% confluent HEK-293T cells with diluted AAV solution, followed by double transfection with pHelper Vector and AAVpro® Packaging Rep-Cap Plasmid (AAV2), and harvesting AAVs as described previously and throughout this example 1 description. DNA is extracted from prepared AAV solutions, the coding sequences of the Synthetic Helper Gene Products are amplified by PCR, purified by gel electrophoresis and gel DNA extraction, and then sequenced using NovaSeq PE150.

[00582] NGS analysis was performed using standard workflows. Briefly, we began with initial preparation and indexing, where each library's NGS region was formatted into a FASTA file. Bowtie2 or BWA was used to build indexes from these files, enabling alignment of sequencing reads to reference sequences. This step was repeated for each unique library in the experiment. Quality control and trimming followed, with raw sequencing reads processed through fastp for quality checks. This tool trimmed low-quality sequences and removed adapters, filtering out poor-quality data. A quality score threshold of 30 was set to ensure high data integrity. Next, the FLASH tool was employed to merge paired-end reads from the cleaned data, creating consensus reads and improving confidence in the read. Alignment was then performed using Bowtie2 or BWA (with BWA being preferred) to align sequencing reads to reference sequences, resulting in SAM files with alignment results for each library. Postalignment processing involved converting SAM files to BAM format using Samtools for smaller file sizes and faster processing, followed by sorting or indexing of these BAM files for quicker analysis. SHGP quantification was conducted by analyzing SHGP-associated DNA reads in naive and enriched datasets. To enable fair comparison, raw counts were converted to frequencies by dividing by the total number of reads in each library. Custom Python scripts with open-source packages were used to extract read counts for each unique SHGP sequence, and the frequency of each SHGP was calculated as its read count divided by the library's total reads. Finally, enrichment calculation was performed by comparing frequencies of each SHGP in naive vs enriched libraries, indicating the SHGP's impact on viral production. Python and various packages were utilized to perform statistical analysis, calculating p-values and adjusting for multiple testing. This workflow allowed us to quantify the impact of millions of SHGP candidates in viral production.

[00583] The viral titer of the libraries were determined using qPCR. The viral titer for individual SHGP variants was determined with a combination of qPCR and NGS data. The method for determining individual SHGP variant titers in pooled samples combines quantitative PCR (qPCR) and next-generation sequencing (NGS) data. The total viral titer of the pooled sample is quantified using qPCR (VG/ml), while NGS determines the frequency of each SHGP variant. The VG/ml for each variant is determined by multiplying the total pool VG/ml by the ratio of the variant's frequency to the average frequency. We can then use this viral titer to determine a fold change in viral titer by dividing it by the reference titer (the VG/ml of the pooled sample). The VG/ml of each variant is validated by comparing the fold difference in VG/ml of a particular variant’s viral titer to its enrichment score (i.e. to confirm that changes in viral titer for a given SHGP within a round agree with changes in frequency between rounds). We additionally apply statistical filtering, considering only variants with p- values less than 0.0001. Using these methods, we identified thousands of SHGPs that increased viral titer and were highly enriched with very low p-values.Fig. 16. Provides a volcano plot that provides and exemplary overview the enrichment profile of SHGP libraries, in the case of Fig. 16a NLH Nanobody SHGP library derived from SEQ ID NO: 216 is shown. The diversity of libraries can be intuitively visualized in Fig. 17, which illustrates a scatter plot of various antibody-like SHGPs that have been clustered by similarity and similar plots were generated for all other SHGP libraries. Fig. 18 provides additional visualization of the diversity comprised by the library, further hinting at sequence-function relationships with certain residues being strongly enriched in Fig. 18 A. Fig. 19 provides additional support for the diversity of within the library, by illustrating the degree of similarity (and difference) between representative library members (as determined by clustering analysis) from within the same library. Together, the methods described within this example were used to demonstrate expedient methods of SHGPlibrary construction, functional enrichment, and functional analysis for developing antibody-like SHGPs. Similarl/equivalent, methods were also used to develop engineered transcription factor based SHGPs. The diversity of 5 exemplary transcription factor based SHGP variations is provided in Figs. 20-24, each providing both a high level view of the sequence landscape with a t-SNE plot (Figs. 20-24 Part A) and a more granular analysis of how specific mutations affect protein function with a DMS plot (Figs. 20- 24 Part B). From this diversity, we were able to identify a number of particularly high- performance SHGPs for both antibody-like protein SHGPs and engineered transcription factor SHGPS.

[00584] Figs. 25-33 provide data to further illustrate the diversity of SHGP libraries (A) as well as the functional diversity contained therein (B). We determined the viral titer of the top 20 most highly enriched SHGP for each category (Figs. 25-33 Part C). For antibody-like SHGPs, we provide 80 representative examples (SEQ ID NOs: 35-114) that were generated from two different scaffolds (SEQ ID NOs: 215-216) that mediate an at least 5-fold increase in viral titer. Figs. 25-28 Part C provide SHGP viral titer measurements compared to the reference for antibody -like SHGPs, while Fig. 35 demonstrates that SHGPs can increase biological titer by at least 10-fold. For engineered transcription factor SHGPs, we provide 100 representative examples (SEQ ID NOs: 115-214) that were generated from five different scaffolds (SEQ ID NOs: 217-220) that mediate an at least 48 fold increase in viral titer, with some variants achieving multi order of magnitude increases. Figs. 29-33 Part C provide SHGP viral titer measurements compared to the reference for antibody-like SHGPs.

[00585] Reagents and materials used in this Example.

[00586] AAVpro 293 T human embryonic kidney cell line (HEK-293T) was obtained from Takara Bio. Fetal Bovine Serum (FBS), Dulbecco’s modified Eagle medium (DMEM) and the penicillin/streptomycin cocktail were obtained from Gene see Scientific. pAAV-CMV Vector, pAAV-ZsGreenl Vector, AAVpro® Packaging Rep-Cap Plasmid (AAV2), pHelper Vector were obtained from Takara Bio. AAV Extraction Solution A and AAV Extraction Solution B were obtained from Takara Bio. pTargeT™ Mammalian Expression Vector System was obtained from Promega. pMiniT Vector, NEB® PCR Cloning Kit and Golden Gate Cloning Kit were obtained from New England Biolabs. EcoRI-HF, BamHI-HF, T4 DNA Ligase, NEB 10-Beta Electrocompetent Cells, and NEB Stable Competent Cells were obtained from New England Biolabs. JetOptimus transfection reagent was obtained from Polyplus.

[00587] Generation of Library peptide library.

[00588] Production of Synthetic Helper Gene Product library was performed using standard molecular cloning strategies: Molecular Cloning: A Laboratory Manual, 4th edition, 2012.

[00589] The coding sequence of Synthetic Helper Gene and eGFP reporter protein were synthesized by Integrated DNA Technologies with EcoRI and BamHI restriction sites flanking the coding sequence. These fragments were cloned into pMiniT Vectors as described in the NEB protocol (Ligation Protocol for NEB PCR Cloning Kit, NEB). To monitor enrichment of AAV-enhancing constructs in the population of host cells, a GFP reporter was incorporated into AAV capsids. A T2A peptide sequence (linker) followed by eGFP reporter coding sequence was inserted prior to the STOP codon of the Synthetic Helper Gene coding sequence using Golden Gate Assembly. Different lengths of Synthetic Helper Gene Product were generated by saturation mutagenesis of the Synthetic Helper Gene2A-eGFP pMiniT Vector using Golden Gate Assembly (Golden Gate Assembly Protocol for NEB Golden Gate Assembly Kit, NEB), followed by transformation into NEB 10-beta Electrocompetent cells as described by the NEB protocol (Electroporation Protocol (C3020), NEB).

[00590] Plasmids were prepared from the resulting transformants and the various length Synthetic Helper Gene-2A-eGFP encoding DNA fragments were then digested out of the pMiniT vector using EcoRI-HF and BamHI-HF and ligated downstream of the ITR (SEQ ID NO: 8-9) flanked constitutive CMV promoter of the pAAV-CMV vector using the EcoRI and BamHI restriction sites before transformation into NEB Stable Competent cells as described in the NEB protocol (High Efficiency Transformation for NEB® Stable Competent E. coli (C3040H), NEB). Plasmids prepared from the resulting transformants were used in AAV production.

[00591] Multiple Synthetic Helper Gene Product libraries were created, with different amino acids lengths, architectures (e.g. nanobody-CDR3 libraries, disordered region peptide fusions) and different numbers of amino acids targeted for mutagenesis. Amino acids located in the region of the peptide to be mutated were randomized using saturation mutagenesis with NNK degenerate codons and the DNA constructed with oligos purchased from IDT. In cases of engineered transcription factor SHGP libraries, the entire peptide coding sequence was mutagenized using error-prone PCR. [00592] Individual Synthetic Helper Gene of interest were PCR amplified from viral AAV DNA, purified by gel electrophoresis and cloned into the pTargeT™ Mammalian Expression Vector System as described in the Promega protocol (pTARGET™ Mammalian Expression Vector System, product # Al 410, Promega). Sanger sequencing was used to determine the identities of the individual Synthetic Helper Gene Products encoded on each vector.

[00593] AAV production and purification.

[00594] 50-70% confluent HEK-293T cells (~10^A6 total cells) grown in DMEM supplemented with 5% FBS were triple transfected with pHelper Vector, pAAV-CMV Vector containing Synthetic Helper Gene-2A-eGFP payload, and AAVpro® Packaging Rep-Cap Plasmid (AAV2) in a 1 : 1 : 1 molar ratio normalized to the plasmid size using JetOptimus Transfection Reagent in 100mm TC treated petri dish.

[00595] 24 hours after transfection, a medium change to DMEM with 2% FBS was performed. On day 3 post-transfection, cells were collected and processed. eGFP expression in packaging HEK-293T cells was quantified using a Tali Image Based Cytometer (Invitrogen). Cells were lysed in an acidic buffer (AAV Extraction Solution A), the homogenates were cleared from debris by centrifugation, and the pH was neutralized using HEPES buffer (AAV Extraction Solution B).

[00596] Subsequent rounds of Synthetic Helper Gene Product enrichment were carried out by transducing 50-70% confluent HEK-293T cells (~10^A6 total cells) grown in DMEM supplemented with 5% FBS with a 1 : 10 to 1 : 10000 dilution of prepared AAV solution from above in DMEM with 5% FBS. Cells were double transfected with pHelper Vector and AAVpro® Packaging Rep-Cap Plasmid (AAV2) in a 1 :1 molar ratio normalized to the plasmid size using JetOptimus Transfection Reagent in 100mm TC treated petri dish. AAVs were harvested as described above.

[00597] AAV Physical Titer Measurement.

[00598] Primers binding the eGFP reporter protein were used to measure the virus titer with quantitative polymerase chain reaction (qPCR). Before releasing the viral DNA from the particles, all extra-viral DNA was removed by digestion with DNase I. Then, the viral DNA was released by alkaline lysis. The qPCR was performed using the PowerUp™ SYBR™ Green Master Mix (Applied Biosystems), and primers against the eGFP reporter protein obtained from Integrated DNA Technologies. The extracted viral DNA and a serial dilution of a plasmid containing eGFP as a standard were measured using the CFX96 Touch Real-Time PCR Detection System and the CFX Maestro Software (Bio-Rad, Hercules, CA). [00599] Biological Viral Titer Measurement

[00600] HEK-293T were reseeded in a 24 well tissue culture treated plate at a density of 1-2* 10^A4 cells/well in 0.5mL of DMEM with 10% FBS. Cells were cultured overnight. 10-fold serial dilutions of prepared AAV2 particle solutions using DMEM with 10% FBS were used to infect the HEK-293T cells. Dilutions ranged from 1 : 10 to 1 : 10000. On Day 3 after infection, cells were detached using Trypsin/EDTA and analyzed for eGFP expression by image-based cytometry.

[00601] Sequencing

[00602] DNA is extracted from prepared AAV solutions of interest as described above. The coding sequences of the Synthetic Helper Gene Products were amplified by PCR. The resulting product is purified by gel electrophoresis and gel DNA extraction before being sequenced. (NovaSeq PE150).

[00603] Synthetic Helper Gene validation

[00604] 50-70% confluent HEK-293T cells (~10^A6 total cells) grown in DMEM supplemented with 5% FBS were quadruple transfected with pHelper Vector, pAAV-ZsGreenl Vector, AAVpro® Packaging Rep-Cap Plasmid (AAV2), and pTargeT Mammalian Expression Vector with cloned individual Synthetic Helper Gene of interest in a 1 : 1 : 1 : 1 molar ratio normalized to the plasmid size using JetOptimus Transfection Reagent in 100mm TC treated petri dish. AAV preparation is conducted as described above. Biological viral titer measurement is performed as described above.

[00605] Example 2. AAV DNA barcoding-based discovery of SHGPs.

[00606] This example illustrates DNA barcoding-based discovery of SHGPs that improve AAV production and in vivo performance.

[00607] It may be desirable to implement an approach that completely removes the Synthetic Helper Gene Product sequence from the transgene plasmid during enrichment, allowing for discovery of Synthetic Helper Gene Products (SHGP) that enhance packaging of therapeutically relevant payloads or discovery of Synthetic Helper Gene Products that may be too large to fit into the AAV ITRs (SEQ ID NO: 8-9). Such an approach provides a good way to perform in vivo discovery, where it would be undesirable to transduce a Synthetic Helper Gene into a living animal model. This can be accomplished through use of a DNA barcodingbased approach. A barcoding based approach provides for a powerful screen, which allows for the rapid identification of viral production-enhancing Synthetic Helper Gene Products. This approach can be visualized in Fig. 6. Fig. 3 exemplifies the application in an in vivo discovery context. Fig. 12 exemplifies how SHGP-associated barcodes are quantified after transduction. [00608] In this approach, a DNA barcode is placed into the mobilizable portion of the plasmid between the ITRs (SEQ ID NO: 8-9), while the Synthetic Helper Gene is encoded on the vector backbone. In this arrangement, the Synthetic Helper Gene Product sequence will be expressed and influence host cell physiology, but the SHGP sequence will not be packaged into AAVs. The Synthetic Helper Gene Product’s impact on viral activity can be determined by its associated, mobilizable DNA barcode, which is packaged into AAVs. It is known in the art how to create DNA barcodes and there are a variety of approaches. Those skilled in the art will recognize that many different DNA barcode designs can be used and there is an extensive body of literature available. Preferably, DNA barcode libraries have a greater diversity than the SHG library and the higher the diversity the better in order to prevent variant identifier clashes. Two examples of how to implement DNA barcoded Synthetic Helper Gene Product AAV libraries are provided below.

[00609] A plasmid containing a DNA barcode is flanked by two ITRs (SEQ ID NO: 8- 9), optionally with an eGFP, luciferase, or therapeutic gene payload. Also on the plasmid, but outside of the ITRs is a Synthetic Helper Gene Product coding sequence.

[00610] Packaging cells are transfected with the DNA library configured to produce a library of Synthetic Helper Gene Products. This SHGP library, along with Rep/Cap and helper plasmids for AAV production (if components needed for packaging are not already supplied in the cell), are transfected into packaging cells.

[00611] The Synthetic Helper Gene Product coding sequence is configured not to be packaged by AAV. Instead, an ITR-flanked DNA barcode is associated with the Synthetic Helper Gene Product. This barcode, when separated from its associated Synthetic Helper Gene Product DNA sequence (e.g. packaged into a viral particle) can be used to identify its associated Synthetic Helper Gene Product (i.e. that was on the same plasmid). The Synthetic Helper Gene Product encoding DNA may also include additional DNA payload elements: fluorescent proteins (eGFP), reporter enzymes (luciferase), therapeutic genes, and more. Elements required for viral production can be supplied via triple transfection of a rep-cap plasmid (pRC) and a helper plasmid (pHelp). Alternatively, these elements can also be expressed from stable genomic integrations of the genes, helper viruses, and so forth.

[00612] Viral packaging occurs. DNA encoding Synthetic Helper Gene Products is associated with a DNA barcode. The DNA barcode is operably linked to packaging sequences (e.g., flanked by two ITRs (SEQ ID NO: 8-9)) which will cause the DNA barcode to be packaged into an AAV. The Synthetic Helper Gene Product, being simultaneously expressed during packaging, is also present during packaging and exerts its biological effect. However, the Synthetic Helper Gene Product coding sequence is not flanked by ITRs and is not packaged into viral particles. Instead, the DNA barcode is used to identify the associated Synthetic Helper Gene Product.

[00613] In this way, Synthetic Helper Gene Products that enhance AAV production will enhance the packaging of their associated DNA barcodes into AAV particles (or increase TU:VG ratios of particles harboring the associated DNA barcodes) and the DNA barcode inside of the AAV particle can be used to identify the Synthetic Helper Gene Product that was present (e.g., associated with the DNA barcode) during the packaging process.

[00614] In contrast, Synthetic Helper Gene Products that reduce AAV production will reduce the efficiency of their associated DNA barcodes from being packaged into AAV particles (or reduce the TU:VG ratios of particles harboring the associated DNA barcodes). In this way, a Synthetic Helper Gene Product’s impact on viral production can be determined by analysis of AAVs harboring the associated DNA barcode.

[00615] Then, the AAV-barcode library is harvested. Each barcode-DNA containing AAV contains a barcode that corresponds to a Synthetic Helper Gene Product that was present during viral packaging. This provides a way to link the quantity or infectiousness of AAVs containing a particular barcode with Synthetic Helper Gene Product that was present during viral packaging.

[00616] The viral titer of the library is preferentially determined at this point to determine the appropriate dosage for transduction in the next step. Too much viral material leads to toxicity. Too little viral material leads to under-sampling of DNA barcodes. We aim for about 1E4-1E5 AAV viral particles per cell with AAV2, but this will be different for different serotypes. 1E5 VG per cell typically results in 10-100 transducing units in AAV2 (this is AAV serotype and packaging methodology dependent).

[00617] These viral particles can then be transduced into cells, for example in vitro or in vivo (e.g., via tail vein injection of 1E13 VGs). It is important to note that the barcode approach is important for the in vivo discovery approach, where it is much less practical to configure the cells of a living animal for viral production. That is, because cells in an animal are not suitable as packaging cells, an approach that does not rely on direct amplification of Synthetic Helper Gene Product viral DNA is required. And for this problem, the exemplified Synthetic Helper Gene Product barcoding approach provides a solution because the barcodes can be easily analyzed by collecting the DNA of transduced animal cells. Transduction can optionally be monitored by observing the luminescence or fluorescence of a transduced payload if configured to allow it. [00618] After a certain number of days, viral DNA is isolated from the cells and subjected to NGS. The naive DNA plasmid library and the remainder of the non-transduced viral library is also NGS-ed. The number of days depends on the particular experiment, but in general, it is at least 5 days. However, it can be preferable to wait for months, depending on the animal model, if desiring to identify highly stable transduction events.

[00619] The sequencing data can then be analyzed to determine which Synthetic Helper Gene Products (indicated by corresponding DNA barcodes) are desirable. The relative frequency of different barcodes (corresponding to different Synthetic Helper Gene Products) is used to determine which Synthetic Helper Gene Products enhanced viral titer and/or infectivity (e.g. by exerting their effect during packaging).

[00620] By analyzing the DNA barcodes associated with specific Synthetic Helper Gene Product sequences and determining enrichment, the functional impacts of Synthetic Helper Gene Product library members can be easily quantified. NGS-based analysis of the DNA barcodes provides the necessary information to identify Synthetic Helper Gene Products that improve in vivo performance, even in the absence of Synthetic Helper Gene Product DNA sequence.

[00621] Assuming that each unique barcode sequence has already been associated with a specific Synthetic Helper Gene Product sequence (e.g., through gene chip synthesis described above), barcode sequences can be mapped to Synthetic Helper Gene Product identity (e.g., using a python dictionary) using the following steps:

[00622] 1) Perform standard NGS analysis and quality control to make a list of DNA reads from raw data files.

[00623] 2) Count the number of occurrences or DNA barcode reads associated with each unique Synthetic Helper Gene Product in the naive, AAV packaged, and post-selection library data files. This information provides the initial quantitative measure of the representation of Synthetic Helper Gene Product sequences associated with each barcode.

[00624] 3) Normalize the read counts within each library to allow for meaningful comparisons between the libraries. While there are many approaches, we typically use frequency which is the number of reads for a particular Synthetic Helper Gene Product divided by the total number of reads for all Synthetic Helper Gene Products.

[00625] 4) Calculate the enrichment of each Synthetic Helper Gene sequence by comparing its frequency in the post-selection library to its frequency in the naive library and/or packaged library. [00626] 5) The fold change or enrichment ratio quantifies the extent to which a

Synthetic Helper Gene Product sequence has become more or less represented in the library as a result of the selection process.

[00627] 6) A fold change greater than 1 indicates an enrichment, while a fold change less than 1 suggests a depletion.

[00628] 7) Comparing enrichments across all three conditions, naive, packaged, and transduced, provides insight not only into impacts of Synthetic Helper Gene Products on in vivo AAV performance, but also AAV production and titer. This provides a way to simultaneously optimize manufacturability and therapeutic aspects of the material.

[00629] Exemplary ways to implement DNA barcoded Synthetic Helper Gene Product AAV libraries are described below.

[00630] Barcoded Synthetic Helper Gene Product libraries can be built in a variety of ways. One way to implement a barcoded Synthetic Helper Gene library is to use a random DNA barcode (for example a string or random “N” nucleotides long enough to ensure that the diversity exceeds the Synthetic Helper Gene Product library’s diversity by a few orders of magnitude) inside of the ITRs (SEQ ID NO: 8-9) along with a mutagenized Synthetic Helper Gene Product library. In this case, we start off not knowing which Synthetic Helper Gene Products and barcodes are associated. At this point, long-read DNA sequencing technology is used to associate the DNA barcodes with the Synthetic Helper Gene Product DNA library sequences. After this step, a short read sequencing technology can be used to provide the sequencing depth required. This approach is much simpler and cheaper to implement.

[00631] Example 3. Lentivirus production with SHGPs.

[00632] This example illustrates methodologies for Synthetic Helper Gene Product- enhanced lentiviral vector production.

[00633] 1) Cells are seeded at 4-5 x 10^A6 cells per 10-cm plate, 24 hours prior to transfection, to achieve 80-90% confluence at the time of transfection.

[00634] 2) 7.0 pg of lentiviral vector plasmid DNA is diluted in sterile water to a volume of 600 pl along with a Synthetic Helper Gene that expresses a Synthetic Helper Gene Product.

[00635] 3) Titration of Synthetic Helper Gene Product plasmid DNA concentrations, from 0.5 ng to 50 ng, is used in this Example. However, the amount of required plasmid DNA depends on many factors, including the specific Synthetic Helper Gene Product being expressed, the Synthetic Helper Gene (e.g., more efficient Synthetic Helper Gene may need less DNA), and promoter strength (the pCMV promoter driving strong expression is used in this Example).

[00636] 4) Between 4-8 control conditions (depending on number of experimental conditions) are also prepared that are identical to the above, except that no Synthetic Helper Gene Product plasmid was added.

[00637] 5) This DNA is added to a tube of Lenti-X Packaging Single Shots and vortexed to mix.

[00638] 6) The mixture is incubated for 10 minutes at room temperature to allow the formation of nanoparticle complexes, then briefly centrifuged.

[00639] 7) The entire 600 pl of nanoparticle complex solution is added dropwise to the packaging cells, which are then rocked gently to mix.

[00640] 8) Lentiviral packaging. Cells are incubated at 37°C with 5% CO2, initially for

4 hours or overnight, then with an additional 6 ml of fresh complete growth medium for another 24-48 hours.

[00641] 9) Lentiviral supernatants are harvested. They were centrifuged or filtered to remove cellular debris, using a filter of cellulose acetate or polysulfone, not nitrocellulose. [00642] 10) Virus production is verified and can be quantified using Lenti-X GoStix™

Plus or any other standard methodology. Then, the virus can be used to transduce target cells or stored at -80°C.

[00643] 11) Next, for each viral production condition (e.g., different Synthetic Helper

Gene Product packaging conditions, SHGP dosages, and controls), a three order of magnitude serial dilution at 10-fold dilution steps is performed.

[00644] 12) All viral samples are then transduced, harboring a GFP transgene, into fresh

HEK293 cells.

[00645] 13) After 3 days, the transduced cells are collected and analyzed by flow cytometry.

[00646] 14) Finally, the biological titer (flow cytometry) and physical titer (GoStix) are normalized to the no-SHGP control to determine the fold improvements in both viral titer and infectiousness.

[00647] 15) While in this Example Synthetic Helper Gene Products are added in a separate plasmid, the Synthetic Helper Gene can also be supplied from a genomically integrated construct in the packaging cells. Alternatively, Synthetic Helper Gene Products can also be exogenously added.

[00648] Example 4. Cell line growth optimization. [00649] Scaling up stable cell lines is a major challenge of viral vector performance. In the context of AAV, the Rep (SEQ ID NO: 1-4, 10, 12, 14) proteins are known to be particularly cytotoxic, while at the same time indispensable for viral production. Rep proteins (SEQ ID NO: 1-4, 10, 12, 14), particularly Rep78 (SEQ ID NO: 4) and Rep68 (SEQ ID NO: 3), have been found to be cytotoxic, causing cell cycle arrest and apoptosis. This can make it challenging to generate stable cell lines for producing AAV vectors. As such, finding a way to address the cytotoxicity while also maintaining the protein’s function would dramatically improve the scalability of viral production with stable cell lines. This example illustrates how the Synthetic Helper Gene Product viral production technology can be used to optimize the properties of a stable producer cell line for viral production.

[00650] A modified selection system that incorporates elements of the AAV selection system and the lentiviral packaging system is established.

[00651] A lentiviral transgene comprising two components is constructed: a Rep gene encoding a Rep protein (SEQ ID NO: 1-4, 10, 12, 14), preferably under control of an inducible promoter system (to adjust selection stringency) and a multiple cloning site, and preferably a selectable marker (e.g., G418/Geneticin to select for stable genome integration effects).

[00652] A DNA expressed Synthetic Helper Gene Product library operably linked to two or more AAV ITRs (SEQ ID NO: 8-9), optionally including a reporter protein, is generated by standard molecular cloning techniques known to those in the art and exemplified in Fig. 14 (for antibody-like SHGPs) and Fig. 15 (for engineered transcription factor based SHGPs).

[00653] This ITR-Synthetic Helper Gene Product library is then inserted into the lentiviral multiple cloning site. The resulting lentiviral transfer vector plasmid DNA now contains a Synthetic Helper Gene Product library capable of being packaged into AAV particles, a Rep protein (SEQ ID NO: 1-4, 10, 12, 14) expression construct (preferably under inducible expression control), which will create the selective pressure, and preferably a selectable marker, which can be used to select for successful lentiviral transductions. This plasmid, being generated in E. coh. is not under any selective pressure from the Rep protein at this point because the E. coli cell is not a mammalian cell (expression is not toxic).

[00654] Next, lentiviral packaging commences using methods described previously. Because the effector compound for inducing Rep (SEQ ID NO: 1-4, 10, 12, 14) expression is withheld, the impact of Rep, while not eliminated, is mitigated. This is enough to permit lentiviral packaging. The resulting lentiviral particles are harvested. [00655] Next, fresh cells (e.g. HEK293) are prepared and transduced by the lentiviral vectors. The effector compound to induce expression of the Rep protein (SEQ ID NO: 1-4, 10, 12, 14) is added. Additionally, the antibiotic compounds are added to select for cells that were successfully transduced by lentivirus, while killing off those that were not. The result is a cell library in which each cell expresses the cytotoxic Rep proteins at an amount determined by the effector compound concentration as well as expression of a Synthetic Helper Gene Product that may serve to detoxify one or more of the Rep proteins.

[00656] This cell library is then subcultured into a large volume of fresh media and a selection experiment is allowed to proceed. Cells that succumb to Rep mediated cytotoxicity will die or grow slowly. On the other hand, cells that manage to successfully detoxify the Rep protein (SEQ ID NO: 1-4, 10, 12, 14) (e.g., via the Synthetic Helper Gene Product) rapidly multiply and are enriched in the large volume of cell culture. It is also possible that cells mutate the Rep and “cheat.”

[00657] After a certain amount of time, fractions of the cells are collected. These cells are then doubly transfected with plasmids that configure them to start to produce AAV particles. It is critical to note that the Rep protein (SEQ ID NO: 1-4, 10, 12, 14) is not supplied at this step, because it must be supplied on the chromosomal insertion. This selects for a functional Rep protein. The ITR-flanked Synthetic Helper Gene Product sequence on the genome of the cell is then mobilized as the cell’s AAV production environment mobilizes the AAV genome and starts packaging it into viral particles. The viral particles are then harvested and can be optionally sequenced via NGS.

[00658] Finally, the DNA is harvested, and the Synthetic Helper Gene Product library can be reinserted into fresh lentiviral Rep vector for another round of selection. This enrichment scheme can be repeated multiple times, preferably being tracked by NGS. Over multiple rounds of enrichment, Synthetic Helper Gene Products that detoxify Rep are selected for. These Synthetic Helper Genes can then be permanently integrated into the genome, along with Rep and other AAV packaging genes to make a highly productive and scalable stable cell line.

[00659] Example 5. Producing the AAV vector composition of increased viral titer and/or transduction efficiency.

[00660] 1. A 100-mm cell culture dish was reseeded with 4.0 x 10^A6 Hek293T cells in high glucose DMEM culture medium supplemented with 10% FBS.

[00661] 2. One day after plating, the cells (70-80% confluence) were co-transfected with 3.2ug pRC2 which encodes Rep (SEQ ID NO: 1-4, 10, 12, 14) and Cap genes (SEQ ID NO: 5-7, 11, 13, 15), 4.6ug pHelper which encodes Adenovirus helper genes (SEQ ID NO: 16- 23), 2.2ug payload vector, and l-100ng Synthetic Helper Gene Product expression vector (if using). A jetOPTIMUS DNA transfection reagent is used during the transfection protocol (from Polyplus).

[00662] 3. 12-16 hours after transfection, media was replaced with low glucose

DMEM culture medium supplemented with 1% FBS, IX Glutamax, lOmM HEPES, and 0.075% sodium bicarbonate.

[00663] 4. Cells and media were harvested 4 days after transfection and frozen in

ImL aliquots.

[00664] 5. Frozen aliquots were thawed and lysed using AAVMax Lysis Reagent

(Life Technologies, USA, A50520). 100 uL of AAVMax Lysis buffer was added to 900 uL of cell and media. The mixture was incubated for 2 hours while rotating. Debris was pelleted at 5,000xg for 15 minutes. AAV containing supernatant was collected.

[00665] 6. The physical and biological titers of the harvested AAV samples were determined by qPCR and biological viral titer measurement as described in Example 1.

[00666] The physical titer fold improvement was calculated by dividing the physical titer of AAVs packaged in cells expressing a SHGP by the physical titer of cells that are not expressing a SHGP (see Fig. 25- Fig. 33 Parts C for viral titers). Fig. 16 and Fig. 25- Fig. 33 illustrate substantial increases in viral production with respect to the library and for individual variants.

[00667] The fold improvement in biological titer can be estimated by determining the viral titer of SHGPs that are enriched in a round 2 selection (which selects for both packaging yield and transduction/infectious efficiency), but which are negligibly enriched in a round 1 selection (which selects principally for increased yield). pRC2, which encodes the Rep (SEQ ID NO: 1-4, 10, 12, 14) and Cap genes for AAV2, was selected for this experiment because AAV2 is the best characterized AAV serotype at present. However, alternative serotypes may be selected based on several desired characteristics: the cell or tissue types being targeted, the safety profile of the serotype or desired payload, and/or the current manufacturing process, Rep/Cap plasmid sets for additional serotypes are commercially available from numerous sources (e.g., Takara Bio USA Inc and Life Technologies, USA Inc). These plasmids commonly encode the Rep gene from AAV2 and substitute the Cap gene for a custom serotype. The helper plasmid for AAV packaging is compatible with all serotypes.

[00668] AAV replication genes, AAV capsid encoding genes, and AAV helper genes suitable for use in the methods disclosed herein (e.g., for production of adeno-associated virus (AAV) vector composition in host cells) are commercially available, and a skilled person would be able to identify a combination of at least one AAV replication gene, at least one AAV capsid encoding gene and at least one AAV helper gene to be expressed in particular host cells to produce a functional AAV vector composition. For example, Takara Bio USA offers AAVpro plasmid sets for AAV2 (Cat. # 6230), AAV1 (Cat. # 6672), AAV5 (Cat. # 6650), and AAV6 (Cat. # 6651). These sets contain the relevant Rep/Cap plasmid that expresses the Rep gene (SEQ ID NO: 1-4, 10, 12, 14) of AAV2 and the Cap gene of each serotype (e.g., pRC2), the helper plasmid that expresses adenovirus E2A, E4, and VA (e.g., pHelper), and a payload vector (e.g., pCMV). In another example, Life Technologies, USA offers the AAV-MAX Control Plasmids Kit (Cat. # A47672) which consists of an optimized mix of three plasmids needed to produce adeno-associated virus serotype 2 (AAV2)-expressing GFP: pAAV-CMV- eGFP, pAAV-Rep2Cap2, and pHelper. These three plasmids must be used with a HEK293 cell line that stably expresses the adenovirus El gene. The kit contains an optimized mix of the three plasmids, as well as a separate tube of each plasmid, providing users with flexibility to change plasmid ratios if desired. There are a variety of other commercially available AAV plasmid kits.

[00669] In another example, methods for generating a plurality of host cells comprising proteins necessary for production of AAV vector compositions are disclosed in US Pat. No. 10415020 B2 and US Pat. No. 10072250 B2, incorporated by reference. Examples of host cell compositions comprising elements essential for AAV vector formation include host cell having (a) a nucleic acid encoding the Rep protein (SEQ ID NO: 1-4, 10, 12, 14), (b) a nucleic acid encoding the Cap protein (SEQ ID NO: 5-7, 11, 13, 15), and (c) a nucleic acid encoding adenovirus-derived elements (SEQ ID NO: 16-23). These nucleic acids can be inserted into a plasmid or a viral vector as one or more nucleic acid constructs capable of providing the elements, and then the plasmid or the viral vector can be introduced into the host cells. Examples of nucleic acid constructs include a pRC2-mi342 vector and pHelper vector (manufactured by TAKARA BIO Inc.), which are commercially available plasmids. In embodiments where host cells are insect cells, and a baculovirus vector is used for introduction of nucleic acid constructs for (a) - (c), vectors such as Bac-Rep and Bac-Cap may be used. In some embodiments, the nucleic acid encoding the Cap protein encodes not only the Cap protein but also an assembly-activating protein (AAP) necessary for formation of an AAV particle in an open reading frame different from that of the encoded Cap protein (Proc. Natl. Acad. Sci. USA, 2010, Vol. 107, pp. 10220-10225). Examples of host cells for preparing AAV vector composition-producing host cells include mammalian cells such as human, monkey, and rodent cells, and non-limiting examples thereof include cells having high transformation efficiency, such as a 293 cell (ATCC CRL-1573), a 293T/17 cell (ATCC CRL-11268), a 293F cell, a 293FT cell (all manufactured by Life Technologies, USA), a G3T-hi cell (W02006/035829), an S 9 cell (ATCC CRL-1711), and an AAV293 cell (manufactured by Stratagene Corp.) which are all commercially available cell lines for viral production.

[00670] Example 6. Producing the lentivirus vector compositions of increased viral titer and/or transduction efficiency.

[00671] In the methods disclosed herein, a protocol may be used for transfection and lentivirus production with Lenti-X Packaging Single Shots, using pre-aliquoted, lyophilized, single tubes of Xfect™ Transfection Reagent premixed with an optimized formulation of Lenti-X lentiviral packaging plasmids (Takara Bio USA, Inc).

[00672] 1. A 100-mm cell culture dish was seeded with 4.0 x 10^A6 Hek293T cells in high glucose DMEM culture medium supplemented with 10% FBS is used.

[00673] 2. One day after plating, the cells reached 70-80 % confluence.

[00674] a. 7ug of lentivirus payload vector and l-100ng Synthetic Helper Gene

Product expression vector (if using) were prepared in 600uL of pure water.

[00675] b. 600uL of the above mixture was transferred into LentiX Packaging

Single Shot tube and the entire mixture was mixed by vortexing before incubating for 10 minutes at room temperature.

[00676] c. Entire mixture was transferred into 100-mm cell culture dish.

[00677] 3. Media were harvested 3 days after transfection.

[00678] 4. Physical titer is measured using LentiX Go-Stix (Takara Bio USA, Inc).

[00679] 5. Biological titer is measured as described in Example 1.

[00680] 6. Fold improvements in viral titer are calculated as described in Example

1.

[00681] The Takara Bio USA Lenti-X Packaging Single Shots (Integrase Deficient)

(Cat. # 631278) was used in this Example because it provides a one-step production method to generate high-titer lentivirus. Each tube contains premeasured, lyophilized Xfect Transfection Reagent and an optimized formulation of lentiviral packaging plasmids. The integration deficient pseudotype was chosen for safety. The choice of pseudotype may be based on a variety of priorities including tissue tropism, cytotoxicity concerns, safety, and/or manufacturing efficiency.

[00682] Lentivirus essential genes (e.g., at least one lentiviral gag gene (SEQ ID NO: 26), at least one lentiviral pol gene (SEQ ID NO: 25), at least one lentiviral rev gene, and at least one lentiviral-compatible env gene (SEQ ID NO: 27)) suitable for use in the methods disclosed herein (e.g., for production of lentivirus vector composition in host cells) are commercially available, and a skilled person would be able to identify a combination of at least one lentiviral gag gene (SEQ ID NO: 26), at least one lentiviral pol gene (SEQ ID NO: 25), at least one lentiviral rev gene (SEQ ID NO: 24), and at least one lentiviral-compatible env gene (SEQ ID NO: 27) to be expressed in particular host cells to produce a functional lentivirus vector composition. For example, there are a variety of commercially available lentivirus plasmid kits. Takara Bio USA offers VSV-G Lenti-X Packaging Single Shots (Cat. # 631276) as well as an envelope free Lenti-X Packaging Single Shots (Cat. # 631294) where the user can use a custom envelope plasmid. In another example, complete description of a lentiviral packaging protocol with links to specific sequences on Addgene and references to nucleic acid sequences can be found in Wickersham IR, et al., Lentiviral vectors for retrograde delivery of recombinases and transactivators. Cold Spring Harb Protoc. 2015 Apr l;2015(4):368-74. In yet another example, production of lentivirus vector composition in host cells is described in US Pat. No. 6207455 Bl, US Pat. No. 9057056 B2 and US Pat. No. 9102943 B2, incorporated herein by reference. Examples of host cell compositions comprising elements essential for production of lentivirus vector composition include packaging cell lines such as PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 or GP+envAM-12 (US Pat. No. 5,278,056), or Psi-Crip (Proc. Natl. Acad. Sci. USA, 85:6460-6464 (1988)). In some embodiments, the at least one lentiviral-compatible env gene is an envelope gene encoding a glycoprotein from an enveloped virus. In one particular embodiment, at least one env gene encodes the protein comprising amino acid sequence set forth in SEQ ID NO: 27.

[00683] In some embodiments, for preventing appearance of a replication competent lentivirus particle, a gag-pol gene and an env gene are not located in proximity in a host cell used in the disclosed methods. For example, it may be preferable to use a host cell having a gal-pol gene and an env gene integrated at different positions on a chromosome, or a host cell having a plasmid containing a gag-pol gene and another plasmid containing an env gene.

[00684] The env gene is not limited to one encoding an envelope protein derived from the same virus as the lentivirus vector composition to be produced. In some embodiments, a host cell for pseudotyped packaging which has an env gene derived from a heterologous virus is used in the disclosed methods. For example, an env gene derived from Moloney murine leukemia virus (MoMLV), vesicular stomatitis virus (VSV), gibbon ape leukemia virus (GaLV), or a gene encoding a protein that can function as env can be used as the env gene. [00685] In some embodiments, a lentivirus vector composition comprises human immunodeficiency virus (HlV)-derived vector, feline immunodeficiency virus (FlV)-derived vector, or simian immunodeficiency virus (SlV)-derived vector. In some embodiments, lentivirus vectors (including HIV, SIV, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV)) depend on several viral regulatory genes in addition to the structural gag-pol-env genes for efficient intracellular replication. Lentiviruses use more complex strategies than classical retroviruses for gene regulation and viral replication, with the packaging signals apparently spreading across the entire viral genome. These additional genes display a web of regulatory functions during the lentiviral life cycle. For example, upon HIV-1 infection, transcription is up-regulated by the expression of Tat through interaction with an RNA target (TAR) in the LTR. Expression of the full-length and spliced mRNAs is then regulated by the function of Rev which interacts with RNA elements present in the gag region and in the env region (RRE) (S. Schwartz et al., J. Virol., 66: 150-159 [1992]). Nuclear export of gag-pol and env mRNAs is dependent on the Rev function. In addition to these two essential regulatory genes, a list of accessory genes, including vif vpr, vpx, vpu, and nef are also present in the viral genome and their effects on efficient virus production and infectivity have been demonstrated, although they are not absolutely required for virus replication (K. and F. Wong- Staal, Microbiol. Rev., 55: 193-205 [1991]; R. A. Subbramanian and E. A. Cohen, J. Virol. 68:6831-6835 [1994]; and D. Trono, Cell 82:189-192 [1995]).

[00686] In some embodiments, for the construction of replication-defective lentiviral vectors, the attenuated HIV-1 constructs are used. In one embodiment, the expression vector can synthesize all viral structural proteins but lacks the packaging signal function (“pHP”), includes a strong promoter (yet preferably not a native HIV-1 LTR), the gag-pol gene, the RRE element and the rev gene. In some embodiments, an expression construct (pHP-1) which contained a modified 5' HIV-1 LTR, a novel major splice donor site based on RSV splice sequences, the entire gag-pol-env, vif, vpr, vpu, tat, and rev genes, a selectable gpt marker gene, and an SV40 polyadenylation signal are used as disclosed in US 6207455 BL [00687] Example 7. Producing a lentivirus vector composition of increased viral titer and/or transduction efficiency by using a Synthetic Helper Gene Product.

[00688] Producing highly infectious lentiviral particles at scale, similar to AAV, presents significant challenges, particularly when using recombinant systems with numerous modifications and differences from wild-type (WT) systems. These differences can lead to deoptimizations that are not entirely understood. For example, vectors that retain certain elements, such as VSV-G envelope glycoprotein (SEQ ID NO: 27) or accessory genes like Vpr and Vif, can be toxic to cells. Inactivation or deletion of these genes can reduce cellular toxicity but may negatively impact transgene expression. Achieving an optimal balance between viral replication, transgene expression, and cellular toxicity remains a crucial challenge in the development of safe and effective lentivirus-based vectors for clinical use. Consequently, there is a need to improve the performance and productivity of lentiviral packaging systems while maintaining an exceptional safety profile.

[00689] The same basic approach described for AAV can be applied to lentivirus despite differences in their distinct lifecycles. Lentivirus is a member of the Retroviridae family, a group of enveloped RNA viruses with a unique replication cycle that involves reverse transcription of their RNA genome into DNA, followed by integration of the DNA into the host cell's genome. Example members include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). These integrating RNA viruses can infect a broad range of human and animal cells.

[00690] The lentiviral genome consists of several genes, including gag, pol, and env, which encode structural proteins, enzymes, and regulatory proteins. Key coding genes include gag (encoding matrix, capsid, and nucleocapsid proteins), pol (encoding reverse transcriptase, integrase, and protease), and env (encoding envelope glycoproteins gpl20 and gp41). These genes play essential roles in viral replication, assembly, gene expression, immune evasion, and interaction with the host cell.

[00691] The viral RNA contains various elements, including promoters, terminators, and other regulatory sequences, that control viral gene expression and replication. The genome also contains regions that are transcribed into non-coding RNAs, which may have regulatory functions. The viral RNA is packaged into nucleocapsids, which are protected by the capsid and envelope proteins.

[00692] Lentiviral replication involves reverse transcription of the viral RNA into DNA, which then integrates into the host cell genome. This integrated provirus serves as a template for viral gene expression and production of new viral particles. To create replication-defective lentiviral vectors, essential genes required for viral replication are removed from the transfer vector and provided in trans by the packaging cell line or plasmid DNA. This prevents the emergence of replication-competent viral particles.

[00693] The capsid and envelope are essential for the replication and spread of lentiviruses. The capsid protects the viral RNA from the host's immune system and ensures the virus can enter and infect cells. The envelope glycoproteins, gpl20 and gp41, play crucial roles in various functions necessary for the virus to replicate and spread, including attachment to the host cell, entry into the host cell, uncoating of the capsid, reverse transcription of the viral RNA, integration into the host genome, assembly of new virions, and regulation of the host immune response.

[00694] Recombinant lentiviral systems have been developed to enable the packaging of genes into lentiviral particles, which can then be transferred to cells through infection with these recombinant lentiviral particles. A variety of recombinant lentiviral packaging systems, including 2nd, 3rd, and 4th generation systems, have been designed to balance safety and productivity. Each generation aims to minimize the possibility of generating replication- competent lentiviruses while maintaining efficient gene delivery.

[00695] Although there are differences among these systems, the general concept involves dividing the single genome of the wild-type (WT) lentivirus into multiple plasmids to prevent the emergence of replication-competent viral particles. Recombinant systems typically comprise a transfer vector, also known as an expression plasmid (containing the desired genes to be packaged into the recombinant viral vector, such as GFP), in conjunction with one or more packaging plasmids that incorporate envelope genes and other essential genes required for viral packaging, including gag, pro, pol, vpr, tat, rev, env, and others.

[00696] Design and production of lentiviral vectors is known in the art: (Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases, 2021; Clinical use of lentiviral vectors, 2017; New developments in lentiviral vector design, production and purification, 2013; Lentiviral Vector Bioprocessing, 2021; Retroviral Vectors for Cancer Gene Therapy, 2016; Concise review on optimized methods in production and transduction of lentiviral vectors in order to facilitate immunotherapy and gene therapy, 2020).

[00697] Examples of elements that may be required in certain embodiments for lentiviral vector production include:

[00698] 1) Structural Proteins - Core Structure Formation

[00699] a) Gag (Encoded by gag gene): The Gag protein is a precursor polyprotein that plays a central role in the formation of the viral core structure. Gag undergoes proteolytic processing to generate matrix protein (MA), capsid protein (CA), and nucleocapsid protein (NC). MA is responsible for membrane binding and targeting Gag to the plasma membrane, CA forms the conical capsid structure that encloses the viral RNA and proteins, and NC binds tightly to the viral RNA, protecting it from digestion by nucleases.

[00700] b) MA (Matrix Protein): The matrix protein lies beneath the viral envelope and associates with the inner leaflet of the host cell membrane. It provides structural support to the virion and plays a role in the assembly and release of new virions. [00701] c) Nucleocapsid (NC): Binds tightly to the viral RNA, protecting it from digestion by nucleases.

[00702] 2) Structural Proteins - Capsid Assembly and Stability

[00703] a) Capsid protein (CA): Forms the conical capsid structure that encloses the viral RNA and proteins. It plays a crucial role in capsid assembly and stability, ensuring the integrity of the viral core. CA interacts with both viral and host factors to orchestrate proper capsid formation during viral assembly.

[00704] 3) Structural Proteins - Envelope

[00705] a) Env (Encoded by env gene): Encodes envelope glycoproteins (gpl20 and gp41) responsible for viral entry into target cells. gpl20 is involved in binding to the host receptor (CD4) and co-receptor (CCR5 or CXCR4), initiating the viral entry process. gp41 mediates fusion of the viral and host cell membranes, allowing entry of the viral core into the target cell.

[00706] 4) Replication Proteins - Pol (Encoded by pol gene): Encodes enzymes essential for viral replication.

[00707] a) Reverse Transcriptase (RT): RT is a multifunctional enzyme responsible for the conversion of the viral RNA genome into DNA through reverse transcription. It possesses both RNA-dependent DNA polymerase activity and RNase H activity, which degrades the RNA template within the RNA-DNA hybrid during reverse transcription.

[00708] b) RNase H: RNase H is an endonuclease that specifically degrades the RNA strand of the RNA-DNA hybrid during reverse transcription. This activity is crucial for the synthesis of the viral DNA.

[00709] c) Integrase (IN): IN catalyzes the integration of the viral DNA into the host cell genome. It cleaves the terminal dinucleotides from the viral DNA ends and mediates the strand transfer reaction, inserting the viral DNA into the host chromosomal DNA.

[00710] d) Protease (PR): PR is responsible for the proteolytic processing of the precursor polyproteins into individual functional proteins during viral maturation. It cleaves Gag and Gag-Pol polyproteins at specific sites, leading to the formation of mature viral proteins required for assembly and infectivity.

[00711] 5) Regulatory Proteins:

[00712] a) Tat (Encoded by tat gene): Transcriptional activator that regulates viral gene expression.

[00713] b) Rev (Encoded by rev gene): Facilitates the nuclear export of viral mRNAs, ensuring efficient viral protein synthesis. [00714] 6) Exemplary Accessory Proteins:

[00715] a) Nef (Encoded by nef gene): Modulates immune response, enhances viral replication, and promotes infectivity.

[00716] b) Vpr (Encoded by vpr gene): Induces cell cycle arrest and facilitates nuclear import of the preintegration complex.

[00717] c) Vif (Encoded by vif gene): Counteracts host antiviral protein APOBEC3G, allowing efficient viral replication.

[00718] d) Vpu (Encoded by vpu gene): Enhances virion release and facilitates CD4 degradation.

[00719] e) Vpx (Encoded by vpx gene): Encoded by HIV-2 and SIV, functions in association with Vpr.

[00720] 6) Nucleic Acid Elements:

[00721] a) RNA genome: The viral genome is composed of single-stranded RNA that carries the genetic information of the virus.

[00722] b) Inverted repeats: Flanking the unique segments of the viral genome, these repeats are essential for circularization and replication of the viral genome.

[00723] c) Open reading frames (ORFs): Segments of the viral genome that encode proteins.

[00724] d) Promoters and enhancers: Regulatory elements involved in the control of gene expression.

[00725] e) Transcriptional regulatory elements: Regions of the viral genome involved in the regulation of viral gene expression.

[00726] f) Termination signals: Sequences that signal the end of transcription.

[00727] g) Long terminal repeats (LTRs): Regions at the ends of the viral genome that contain important regulatory elements.

[00728] h) Polyadenylation signal: Sequence that directs the addition of a poly(A) tail to the viral mRNA.

[00729] Exemplary Recombinant lentivirus vector systems.

[00730] A variety of lentiviral systems have been developed, including replication competent, attenuated, and replication incompetent. Recombinant lentiviral systems allow genes to be packaged into lentiviral particles and subsequently transferred to cells through infection with these recombinant lentiviral vectors. Recombinant lentiviral packaging systems come in a variety of forms, attempting to balance safety and productivity. Each system has been designed to reduce the possibility of generating replication-competent lentivirus while maintaining efficient gene delivery. Such recombinant systems comprise a transfer vector, also known as an expression plasmid (which contains the desired genes to be packaged into the recombinant viral vector, such as GFP), along with one or more packaging plasmids that provide the essential genes required for viral replication and packaging.

[00731] In one embodiment, the invention introduces synthetic SHGPs that enhance various aspects of lentiviral vector production, including viral replication, yield, scalability, infectiousness, and decreased cytotoxic effects on host cells. These advancements significantly improve the suitability of lentivirus vectors for applications such as human gene therapy, gene transfer, and other applications in biotechnology and medicine.

[00732] Synthetic Helper Gene Product disclosed herein can enhance production of lentivirus vectors.

[00733] In one embodiment, SHGPs are used to enhance production of lentivirus by an increase infectious titer, defined as the number of infectious units per mL, physical titer, defined as the number of viral particles per mL, or TU:VG ratio, defined as the ratio of transducing units to viral genomes. The embodiment can be described as follows:

[00734] 1) Cell Component: The cell component of this embodiment involves the use of a cell line configured to express the essential lentiviral genes Gag (SEQ ID NO: 26), Pol (SEQ ID NO: 25), and Env (SEQ ID NO: 27). These genes provide the necessary factors for lentiviral replication and packaging.

[00735] 2) Viral Component: The viral component of this embodiment includes a transfer vector carrying a therapeutic gene payload. The transfer vector contains the necessary lentiviral elements, such as the capsid protein (e.g., CA), reverse transcriptase (RT), integrase (IN), and protease (PR), as well as essential regulatory elements (e.g., LTR, packaging signal). The therapeutic gene payload is positioned within the lentiviral genome for efficient packaging and delivery to target cells. The lentivirus is a member of the Retroviridae family, including lentiviral derivatives, hybrids of more than one line, or chimeras of more than one type of virus (e.g., lentivirus-AAV, lentivirus-AdV).

[00736] 3) SHGP embodiment variations:

[00737] a) A SHG is stably integrated into the genome of the packaging cell.

[00738] c) A SHG is added to the transfer vector in such a way that it is not packaged, for example by placing the SHG outside of the terminal repeats (e.g. on a shuttle plasmid) [00739] In another embodiment, SHGPs are discovered using an enrichment-based approach. [00740] A large SHG library is inserted into the transfer vector as a recombinant payload gene. The lentivirus transfer vector bearing the SHGP library is then transfected, along with other packaging plasmids, into the packaging cell. Upon transfection, the plasmids begin to express.

[00741] 1) The packaging cell line generates the necessary materials for recombinant viral production and packaging of the SHGP library variant(s) within the cell.

[00742] 2) Simultaneously, the SHG is expressed from the expression plasmid or transfer vector resulting in the intracellular biosynthesis of Synthetic Helper Gene Product(s) encoded by the SHG variant(s).

[00743] The SHGPs then bind their targets, causing a perturbation in the cell's physiological state.

[00744] 1) If this SHGP-induced cellular perturbation improves the cell’s ability to produce viral particles by any metric (e.g., yield, quality/infectiousness), then the SHGP gene sequence will be more efficiently packaged and will increase its relative representation of the population.

[00745] 2) In contrast, if this SHGP-induced cellular perturbation reduces the cell’s ability to generate viral particles, then the SHGP gene sequence will be less efficiently packaged and will be depleted or eliminated from the population.

[00746] SHGP-induced cellular perturbations may alter several host cell or lentivirus- specific processes, including viral replication, genome synthesis, viral assembly, and egress. The efficiency of these processes can be dramatically altered by the cellular context or perturbations like mutations or changes in gene expression. The resulting library of viral particles will contain a subset of the original SHGP diversity:

[00747] 1) Lentiviral particles harboring SHGP gene sequences that reduced viral packaging are reduced or eliminated from the library (these SHGPs might be antivirals, and indeed might find utility in such applications)

[00748] 2) Viral particles harboring SHGP gene sequences that improved viral packaging are increased or enriched in the library

[00749] 3) The SHGP gene sequences that had no impact on the viral packaging are also reduced due to competition from Viral particles harboring SHGPs gene sequences that improved viral packaging and outcompeted the neutral variants.

[00750] Resulting lentivirus vectors, each harboring one or more Synthetic Helper Gene sequences can then be used to infect fresh packaging cells configured to generate additional lentivirus vectors. [00751] 1) For viral particles to be generated, the genetic sequence encoded by the transfer vector / expression plasmid payload, which contains the SHGP DNA sequence, must be present inside of the packaging cell. This requires that viral particles be functional and able to successfully deliver their genetic payload, which contain the SHGP sequences, into the cell. [00752] 2) SHGP gene sequences in non-functional viral particles (e.g. they interfere with viral production) are eliminated.

[00753] 3) In contrast, viral particles that harbor SHGP gene sequences that previously altered the packaging cell in a way that resulted in enhanced infectivity, quality, payload delivery, or simply a higher population of viral particles with the same infectiousness, will result in the delivery of more of these performance-enhancing SHGP payloads into packaging cells.

[00754] 4) Viral particles that are better able to deliver their SHGP gene sequence payload into the cell (e.g. because there are more of them or because the SHGP enhances viral production) gain a selective advantage and can increase their representation in the population. [00755] This increases the number of cells that will generate viral particles harboring SHGP gene sequences that enhance viral production. In other words: Synthetic Helper Gene Products that enhance viral production enhance the packaging of their own coding DNA into more or better viral particles.

[00756] In summary, SHGP gene sequences that increase infectiousness or viral yield gain a selective advantage and become enriched in the population. SHGP gene sequences that decrease lentivirus production are rapidly depleted. SHGP gene sequences can be isolated and analyzed for their impact on lentivirus production.

[00757] At each step, the SHGP library can be subjected to NGS to understand the composition of the entire library. This provides information about which variants are most likely to be useful for increasing physical and biological titer. SHGP gene sequences that show very high-performance enhancements can be synthesized and tested individually for their ability to increase viral production.

[00758] In addition, after enrichment, the library can be screened directly to identify viral production enhancing SHGPs.

[00759] Example 8. Producing a herpes simplex virus (HSV) vector composition of increased viral titer and/or transduction efficiency by using a Synthetic Helper Gene Product. [00760] In some embodiments, Synthetic Helper Gene Products are used to obtain recombinant HSV vector composition of increasing viral titer and/or transduction efficiency. Essential genes and components for recombinant HSV packaging are known in the art. Similar to AAV, producing highly infectious HSV particles at scale presents significant challenges, particularly when using recombinant systems that have numerous modifications and differences from the wild-type (WT) systems. These differences can lead to deoptimizations that are not entirely understood. For example, vectors that retain the ISHGPO and ISHGP22 IE genes are toxic to cells. Inactivation or deletion of these genes, particularly the ISHGPO gene, can reduce cellular toxicity but may negatively impact transgene expression. Achieving an optimal balance between viral replication, transgene expression, and cellular toxicity remains a crucial challenge in the development of safe and effective HSV-based vectors for clinical use. Consequently, there is a need to improve the performance and productivity of HSV packaging systems while maintaining an exceptional safety profile.

[00761] The same basic approach described for AAV can be applied to HSV despite differences in HSV's distinct lifecycle. Herpes simplex virus (HSV) is a member of the Herpesviridae family, a group of large, enveloped DNA viruses with a lytic-latent replication cycle. Example members include simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human herpesvirus 6 and/or human herpesvirus 7.

[00762] These non-integrating DNA viruses can infect a broad range of human and animal cells. The viral genome consists of over 80 genes and is composed of two unique segments, UL (unique long) and US (unique short), each flanked by inverted repeats that encode critical diploid genes, as well as inverted repeat regions. These inverted repeats are essential for circularization and replication of the viral genome.

[00763] The genome contains a variety of genes encoding viral proteins, including structural proteins, enzymes, and regulatory proteins. Key coding genes include ULI 9 (VP5), UL38 (VP19C), UL18 (VP23), UL26.5 (VP21), UL35 (VP26), UL19 (VP28), UL26 (VP30 and VP35), UL48 (VP16), UL37 (VP19A), UL49 (VP22), UL54 (VP27), UL31 (UL31), and more. These genes play essential roles in viral replication, assembly, gene expression, immune evasion, and interaction with the host cell.

[00764] The viral DNA contains various elements, including promoters, terminators, and other regulatory sequences, that control viral gene expression and replication. The genome also contains regions that are transcribed into non-coding RNAs, which may have regulatory functions. The viral DNA is packaged into nucleocapsids, which are protected by the capsid and tegument proteins. [00765] HSV replication involves temporally regulated expression of its genes in waves, referred to as cascade regulation. This regulation involves the sequential expression of immediate-early (IE) genes, early (E) genes, and late (L) genes. The IE genes are the first to be expressed and function to regulate the subsequent expression of E and L genes. Removal of the essential IE genes ISHGP27 and ISHGP4 renders the virus completely defective and incapable of expressing E genes involved in viral genome replication and L genes functioning in progeny virion assembly. These replication-defective viruses can be grown on complementing cells that express (complement) the missing ISHGP4 and ISHGP27 gene products. After propagation, they can then be used to infect non-complementing cells, where the viral genome resides as a stable nuclear episome, facilitating long-term transgene expression.

[00766] The design of HSV vectors is known for someone skilled in the art: (HSV Recombinant Vectors for Gene Therapy, 2010). While there are differences between the systems, the general concept involves using a replication-defective HSV genome as the transfer vector. The essential genes required for viral replication are provided in trans by the packaging cell line, a helper virus, plasmid DNA, etc. . . This prevents the emergence of replication-competent viral particles.

[00767] The capsid and tegument are essential for the replication and spread of HSV. The capsid protects the viral genome from the host's immune system and helps ensure the virus can enter and infect cells. The tegument plays a crucial role in various functions necessary for the virus to replicate and spread, including attachment to the host cell, entry into the host cell, uncoating of the capsid, transport of the viral DNA to the nucleus, replication of the viral DNA, assembly of new virions, and regulation of the host immune response. Examples of elements that may be required in certain embodiments for HSV vector production include: [00768] Capsid (mostly early genes): VP5 (encoded by ULI 9 gene): Main component of the capsid, involved in the formation and stability of the viral capsid; VP19C (encoded by UL38 gene): Found in the capsid, but its specific function is not well defined; VP23 (encoded by ULI 8 gene): Present in the capsid and important for virus replication and formation of infectious viral particles; VP21 (encoded by UL26.5 gene): Functions in capsid assembly and DNA packaging.

[00769] Other exemplary capsid proteins may include: VP26 (encoded by UL35 gene): Plays a role in capsid assembly and stability; VP28 (encoded by ULI 9 gene): Involved in the assembly and stability of the capsid; VP30 (encoded by UL26 gene): Participates in capsid assembly and packaging of viral DNA; VP35 (encoded by UL26 gene): Contributes to capsid stability and assembly. [00770] Exemplary tegument proteins may include:

[00771] 1) Early genes: VP 16 (encoded by UL48 gene): Involved in the regulation of viral gene expression and host immune response; VP19A (encoded by UL37 gene): Functions in tegument assembly and stability; VP22 (encoded by UL49 gene): Participates in tegument assembly and is important for efficient viral replication.

[00772] 2) Late genes: VP24 (encoded by UL35 gene): Plays a role in tegument assembly and virion egress; VP27 (encoded by UL54 gene): Involved in the regulation of host immune response and viral gene expression; VP39 (encoded by UL26 gene): Required for efficient tegument assembly and stability; UL31 (encoded by UL31 gene): Participates in tegument assembly and virion maturation.

[00773] In certain embodiments, additional tegument components include:

[00774] 1) Lipids: Lipid molecules are present in the tegument and contribute to its structure and stability.

[00775] 2) Carbohydrates: Carbohydrate molecules are found in the tegument and may be involved in interactions with host cells.

[00776] 3) Nucleic acids: Tegument can contain viral and host nucleic acids, which may play roles in viral replication and modulation of host responses.

[00777] In certain embodiments, additional essential regulatory proteins include: ISHGP0 (Infected Cell Protein 0, encoded by the RL2 gene): Functions as a key regulator of viral gene expression, contributing to viral replication and establishment of infection;

ISHGP4 (Infected Cell Protein 4, encoded by the IE175 gene): Essential for viral gene expression, serving as a transcriptional activator of both early and late genes; ISHGP22 (Infected Cell Protein 22, encoded by the US1 gene): Plays a role in regulating viral gene expression, particularly in the control of viral transcription and splicing; ISHGP27 (Infected Cell Protein 27, encoded by the UL54 gene): Essential for viral gene expression and post- transcriptional processing of viral RNA.

[00778] In certain embodiments, essential Nucleic Acid Elements include:

[00779] 1) Terminal Repeat Long (TRL) and Inverted Repeat Long (IRL): Flanking sequences at the ends of the long unique segment (UL) of the HSV-1 genome.

[00780] 2) Internal Repeat Short (IRS) and Terminal Repeat Short (TRS): Flanking sequences at the ends of the short unique segment (Us) of the HSV-1 genome.

[00781] 3) Pac (Packaging Sequence): A specific sequence located within the unique long (UL) region that is recognized by the viral packaging machinery during the encapsidation of viral DNA. [00782] In certain embodiments, additional regulatory elements include regulatory elements involved in gene expression, replication, and genome organization.

[00783] A variety of HSV systems have been developed, including replication competent, attenuated, replication incompetent. Recombinant HSV systems allow genes to be packaged into HSV particles and subsequently transferred to cells through infection with these recombinant HSV vectors. Recombinant HSV packaging systems come in a variety of forms, attempting to balance safety and productivity. Each system has been designed to reduce the possibility of generating replication-competent HSV while maintaining efficient gene delivery. Such recombinant systems comprise a transfer vector, also known as an expression plasmid (which contains the desired genes to be packaged into the recombinant viral vector, such as GFP), along with a packaging cell line that provides the essential genes required for viral replication and packaging.

[00784] SHGPs can be used to improve HSV production. In one embodiment, SHGPs are used to enhance production of HSV by an increase infectious titer, defined as the number of infectious units per mL, physical titer, defined as the number of viral particles per mL, or TU:VG ratio, defined as the ratio of transducing units to viral genomes. The embodiment is described as follows:

[00785] 1) Cell Component: The cell component of this embodiment involves the use of a HEK293 cell line that has been modified to express the essential viral genes ISHGP4, ISHGP22, ISHGP27, and ISHGP47. These genes provide the necessary factors for viral replication and packaging. The modified HEK293 cell line serves as a stable cell line to produce HSV viral vector.

[00786] 2) Viral Component: The viral component of this embodiment includes a transfer vector carrying a therapeutic gene payload. The transfer vector contains the necessary viral elements, such as the capsid proteins (e.g., VP5, VP19C, VP23, VP21) and essential regulatory elements (e.g. TRL/IRL, IRS/TRS, pac sequence). The therapeutic gene payload is positioned within the viral genome for efficient packaging and delivery to target cells. The virus is a member of the Herpesviridae, their derivatives, hybrids of more than one line, or chimeras of more than one type of virus (e.g. HSV-lenti, HSV-AAV, HSV-AdV).

[00787] 3) SHGP embodiment variations:

[00788] a) A SHG is stably integrated into the genome of the packaging cell.

[00789] c) A SHG is added to the transfer vector in such a way that it is not packaged, for example by placing the SHG outside of the terminal repeats (e.g. on a shuttle plasmid) [00790] In another embodiment, SHGPs are discovered using an enrichment-based approach.

[00791] A large SHGP gene sequence library is inserted into the transfer plasmid as a recombinant payload gene. The HSV expression plasmid bearing the SHGP gene sequence library is then transfected, along with other packaging plasmids, into the packaging cell. Upon transfection, the plasmids begin to express the Synthetic Helper Gene Product(s) encoded by the gene sequence variant(s). The packaging cell line generates the necessary materials for recombinant viral production and packaging of the SHGP gene sequence library variant(s) within the cell. The SHGPs then bind their targets, causing a perturbation in the cell's physiological state. If this SHGP-induced cellular perturbation improves the cell’s ability to produce high-quality viral particles by any metric (e.g., yield, quality/infectiousness), then the SHGP gene sequence will be more efficiently packaged and will increase its relative representation of the population. In contrast, if this SHGP-induced cellular perturbation reduces the cell’s ability to generate viral particles, then the SHGP gene sequence will be less efficiently packaged and will be depleted or eliminated from the population. SHGP-induced cellular perturbations may alter several host cell or HSV-specific processes, including viral replication, DNA synthesis, viral assembly, and egress. The efficiency of these processes can be dramatically altered by the cellular context or perturbations like mutations or changes in gene expression. The resulting library of viral particles will contain a subset of the original SHGP diversity:

[00792] 1) Viral particles harboring SHGP gene sequences that reduced viral packaging are reduced or eliminated from the library (these SHGPs might be antivirals, and indeed might find utility in such applications).

[00793] 2) Viral particles harboring SHGP gene sequences that improved viral packaging are increased or enriched in the library.

[00794] 3) The SHGP gene sequences that had no impact on the viral packaging are also reduced due to competition from Viral particles harboring SHGP gene sequences that improved viral packaging and outcompeted the neutral variants.

[00795] Resulting HSV vectors, each harboring one or more Synthetic Helper Gene sequences can then be used to infect fresh packaging cells configured to generate additional HSV vectors. For viral particles to be generated, the DNA sequence encoded on the transfer vector / expression plasmid payload, which contains the SHGP gene sequence, must be present inside of the packaging cell. This requires that viral particles be functional and able to successfully deliver their genetic payload, which contains the SHGP gene sequence, into the cell. SHGP gene sequences in non-functional viral particles (e.g. they interfere with viral production) are eliminated. In contrast, viral particles that harbor SHGP gene sequences that previously altered the packaging cell in a way that resulted in enhanced infectivity, quality, payload delivery, or simply a higher population of viral particles with the same infectiousness, will result in the delivery of more of these performance-enhancing SHGP gene sequence payloads into packaging cells. Viral particles that are better able to deliver their SHGP gene sequence payload into the cell (e.g. because there are more of them or because the encoded SHGPs enhance viral production) gain a selective advantage and can increase their representation in the population. This increases the number of cells that will generate viral particles harboring SHGP gene sequences that enhance viral production. In other words: Synthetic Helper Gene Products that enhance viral production enhance the packaging of their own coding DNA into more or better viral particles.

[00796] Example 9. Producing an Adenovirus (AdV) vector composition of increased viral titer and/or transduction efficiency by using a Synthetic Helper Gene Product.

[00797] In some embodiments, Synthetic Helper Gene Products are used to obtain recombinant adenovirus (AdV) vector composition of increasing viral titer and/or transduction efficiency. Essential genes and components for recombinant adenovirus (AdV) packaging are known in the art. Exemplary genes and components are indicated below. Inverted Terminal Repeats (ITRs) are necessary for the initiation of viral DNA replication and packaging of the AdV genome into viral capsids. AdV packaging signal ( ) is a cis-acting element required for the encapsidation of the viral genome into the viral capsid. AdV early (E) genes are crucial for viral replication and gene expression. AdV El A (SEQ ID NO: 16) and E1B (SEQ ID NO: 17) are involved in the activation of other viral genes and cellular factors necessary for viral replication. AdV E2A (SEQ ID NO: 18) and E2B encode essential components of the viral DNA replication machinery, including the DNA polymerase and the preterminal protein. AdV E4 (SEQ ID NO: 19) contributes to efficient viral DNA replication, late gene expression, and host cell shutoff. AdV late (L) genes encode structural proteins, including capsid proteins (e.g., hexon, penton base, and fiber), core proteins, and other components required for viral assembly and maturation. For recombinant AdV packaging, the transfer vector should include the gene of interest, ITRs , and the packaging signal to facilitate replication and packaging. The packaging cell line should provide the essential AdV genes, such as the El (SEQ ID NO: 16- 17), E2 (SEQ ID NO: 18), and E4 (SEQ ID NO: 19) genes, in trans to support viral replication, gene expression, and assembly of viral particles. By supplying these genes in trans and using a packaging cell line with a deleted El region, the production of replication-defective recombinant AdV particles can be achieved, improving the safety profile of the viral vector for gene therapy applications.

[00798] Producing highly infectious adenoviral particles at scale, similar to AAV and lentivirus, presents significant challenges, particularly when using recombinant systems with numerous modifications and differences from wild-type (WT) systems. These differences can lead to deoptimizations that are not entirely understood. For example, vectors that retain the El A (SEQ ID NO: 16) and E1B (SEQ ID NO: 17) early genes in adenoviral systems can be toxic to cells. Inactivation or deletion of these genes can reduce cellular toxicity but may negatively impact viral production. Achieving an optimal balance between viral replication, transgene expression, and safety remains a crucial challenge in the development of safe and effective adenovirus-based vectors for clinical use. Consequently, there is a need to improve the performance and productivity of adenoviral packaging systems while maintaining an acceptable safety profile.

[00799] The same basic approach described for AAV and lentivirus can be applied to adenovirus despite differences in their distinct lifecycles. Adenovirus is a non-enveloped DNA virus with a linear, double-stranded DNA genome. The Adenoviridae family includes various human and animal adenoviruses with diverse tropisms and pathogenicity.

[00800] The adenoviral genome consists of several genes, including early genes (El A, E1B, E2A, E2B, E3, and E4), late genes (L1-L5), and non-coding elements, which encode structural proteins, enzymes, and regulatory proteins. Key coding genes include hexon (L3 gene), penton base (L2 gene), and fiber (L5 gene) for the viral capsid, and protein VII (LI gene), protein V (L3 gene), and terminal protein (LI gene) for the viral core. These genes play essential roles in viral replication, assembly, gene expression, immune evasion, and interaction with the host cell.

[00801] The viral DNA contains various elements, including promoters, enhancers, inverted terminal repeats (ITRs), and packaging sequences, that control viral gene expression and replication. The viral DNA is packaged into the viral core, which is protected by the icosahedral capsid.

[00802] Adenoviral replication involves the replication of the viral DNA, transcription of viral genes, and assembly of new viral particles. Adenoviral replication involves the replication of viral DNA, transcription of viral genes, and assembly of new viral particles. The early genes (El A, E1B, E2A, E2B, E3, and E4) are involved in viral and cellular gene expression regulation, viral DNA replication, and host immune response modulation, while the late genes (L1-L5) encode structural proteins for the capsid and core assembly. The capsid proteins, including hexon, penton base, and fiber, are essential for the replication and spread of adenoviruses. The capsid protects the viral DNA from the host's immune system and ensures the virus can enter and infect cells. The fiber protein plays a crucial role in binding to the host cell receptor and initiating viral entry.

[00803] Recombinant adenoviral systems have been developed to enable the packaging of genes into adenoviral particles, which can then be transferred to cells through infection with these recombinant adenoviral particles. A variety of recombinant adenoviral packaging systems, including replication competent, attenuated, and replication-deficient, have been designed to balance safety and productivity. Each system aims to minimize the possibility of generating replication-competent adenoviruses while maintaining efficient gene delivery.

[00804] To create replication-defective adenoviral vectors, essential genes required for viral replication are removed from the transfer vector and provided in trans by the packaging cell line or plasmid DNA. This prevents the emergence of replication-competent viral particles. [00805] Although there are differences among these systems, the general concept involves dividing the single genome of the wild-type (WT) adenovirus into multiple plasmids to prevent the emergence of replication-competent viral particles. Recombinant systems typically comprise a transfer vector, also known as an expression plasmid (containing the desired genes to be packaged into the recombinant viral vector, such as GFP), in conjunction with a packaging cell line that provides the essential genes required for viral replication and packaging.

[00806] Design and production of lentiviral vectors is known in the art: Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy, 2013; Adenovirus Biology, Recombinant Adenovirus, and Adenovirus Usage in Gene Therapy, 2021; High-Capacity Adenoviral Vectors: Expanding the Scope of Gene Therapy, 2020; Adenoviral gene therapy, 2002; US20040161848A1 (incorporated herein by reference), Adenoviral vectors and related systems, and methods of manufacture and use).

[00807] Examples of elements that may be required in certain embodiments for adenoviral vector production include:

[00808] 1) Capsid Proteins: Hexon (encoded by L3 gene): Major capsid protein that forms the outer surface of the viral particle and is involved in viral entry; Penton base (encoded by L2 gene): Forms the vertex of the icosahedral capsid and anchors the fiber protein; Fiber (encoded by L5 gene): Projects from the penton base and is responsible for binding to the host cell receptor, initiating viral entry. [00809] 2) Core Proteins: Protein VII (encoded by LI gene): Binds and condenses the viral DNA within the viral core; Protein V (encoded by L3 gene): Contributes to the stability of the viral core; Terminal protein (encoded by LI gene): Covalently attached to the 5' ends of the viral DNA and plays a role in viral DNA replication.

[00810] 3) Early Genes: El A (encoded by El A gene): Involved in the regulation of viral and cellular gene expression, promoting cell cycle progression and viral replication; E1B (encoded by E1B gene): Functions in the inhibition of apoptosis and regulation of cellular gene expression; E2A (encoded by E2A gene): Encodes the DNA-binding protein, which is involved in viral DNA replication and transcription regulation; E2B (encoded by E2B gene): Encodes the viral DNA polymerase, which is essential for viral DNA replication.

[00811] 4) Late Genes: L1-L5 (encoded by L1-L5 genes): Encode structural proteins involved in capsid and core assembly, as well as the stability of the viral particle; E3 (encoded by E3 gene): Encodes proteins that modulate host immune response and facilitate viral egress; E4 (encoded by E4 gene): Encodes proteins involved in viral DNA replication, transcription regulation, and host cell shutoff.

[00812] 5) Exemplary Non-coding Elements: Inverted Terminal Repeats (ITRs): Present at both ends of the viral genome and play a role in viral DNA replication and packaging; Packaging Sequence (y): Located near the left end of the viral genome, it is recognized by the viral packaging machinery during the encapsidation of viral DNA; Promoters and enhancers: Regulatory elements involved in the control of viral gene expression.

[00813] A variety of adenoviral systems have been developed, including replication competent, attenuated, and replication-deficient. Recombinant adenoviral systems allow genes to be packaged into adenoviral particles and subsequently transferred to cells through infection with these recombinant adenoviral vectors. Recombinant adenoviral packaging systems come in a variety of forms, attempting to balance safety and productivity. Each system has been designed to reduce the possibility of generating replication-competent adenovirus while maintaining efficient gene delivery. Such recombinant systems comprise a transfer vector, also known as an expression plasmid (which contains the desired genes to be packaged into the recombinant viral vector, such as GFP), along with a packaging cell line that provides the essential genes required for viral replication and packaging.

[00814] In one embodiment, the present disclosure teaches synthetic SHGPs that enhance various aspects of adenoviral vector production, including viral replication, yield, scalability, infectiousness, and decreased cytotoxic effects on host cells. These advancements significantly improve the suitability of adenoviral vectors for applications such as human gene therapy, gene transfer, and other applications in biotechnology and medicine.

[00815] SHGPs can be used to improve adenoviral production. In one embodiment, SHGPs are used to enhance production of adenovirus by an increase infectious titer, defined as the number of infectious units per mL, physical titer, defined as the number of viral particles per mL, or TU:VG ratio, defined as the ratio of transducing units to viral genomes. The embodiment is described as follows:

[00816] 1) Cell Component: The cell component of this embodiment involves the use of a cell configured to express the essential adenoviral genes El, E2, and E4. These genes provide the necessary factors for adenoviral replication and packaging.

[00817] 2) Viral Component: The viral component of this embodiment includes a transfer vector carrying a therapeutic gene payload. The transfer vector contains the necessary adenoviral elements, such as early genes (El A, E1B, E2A, E2B, E3, and E4), late genes (LILS), and non-coding elements (e.g., inverted terminal repeats (ITRs), packaging sequences). The therapeutic gene payload is positioned within the adenoviral genome for efficient packaging and delivery to target cells. Adenovirus is a member of the Adenoviridae family, and recombinant systems may include adenoviral derivatives, hybrids of more than one serotype, or chimeras of more than one type of virus (e.g., adenovirus-AAV, adenovirus- lentivirus).

[00818] 3) SHGP embodiment variations:

[00819] a) A SHG is stably integrated into the genome of the packaging cell.

[00820] c) A SHG is added to the transfer vector in such a way that it is not packaged, for example by placing the SHG outside of the terminal repeats (e.g. on a shuttle plasmid) [00821] In another embodiment, SHGPs are discovered using an enrichment-based approach. A large SHGP gene sequence library is inserted into the transfer vector as a recombinant payload gene. The adenoviral transfer vector bearing the SHGP gene sequence library is then transfected, along with other packaging plasmids, into the packaging cell. Upon transfection, the plasmids begin to express the encoded Synthetic Helper Gene Product. The packaging cell line generates the necessary materials for recombinant viral production and packaging of the SHGP gene sequence library variant(s) within the cell. The SHGPs then bind their targets, causing a perturbation in the cell's physiological state. If this SHGP-induced cellular perturbation improves the cell’s ability to produce viral particles by any metric (e.g., yield, quality/infectiousness), then the SHGP gene sequence will be more efficiently packaged and will increase its relative representation of the population. In contrast, if this SHGP-induced cellular perturbation reduces the cell’s ability to generate viral particles, then the SHGP expression cassette will be less efficiently packaged and will be depleted or eliminated from the population. SHGP-induced cellular perturbations may alter several host cell or adenoviral- specific processes, including viral replication, genome synthesis, viral assembly, and egress. The efficiency of these processes can be dramatically altered by the cellular context or perturbations like mutations or changes in gene expression. The resulting library of viral particles will contain a subset of the original SHGP diversity:

[00822] 1) Adenoviral particles harboring SHGPs gene sequences that reduced viral packaging are reduced or eliminated from the library (these SHGPs might be antivirals, and indeed might find utility in such applications)

[00823] 2) Viral particles harboring SHGPs gene sequences that improved viral packaging are increased or enriched in the library

[00824] 3) The SHGPs that had no impact on the viral packaging are also reduced due to competition from viral particles harboring SHGPs gene sequences that improved viral packaging and that outcompeted the neutral variants.

[00825] Resulting adenoviral vectors, each harboring one or more Synthetic Helper Gene sequences can then be used to infect fresh packaging cells configured to generate additional adenoviral vectors. For viral particles to be generated, the transfer vector / expression plasmid is present inside of the packaging cell. This requires that viral particles be functional and able to successfully deliver their genetic payload into the cell. SHGP gene sequences in non-functional viral particles (e.g. they interfere with viral production) are eliminated. In contrast, viral particles that harbor SHGP gene sequences that previously altered the packaging cell in a way that resulted in enhanced infectivity, quality, payload delivery, or simply a higher population of viral particles with the same infectiousness, will result in the delivery of more of these performance enhancing SHGP gene sequence payloads into packaging cells. Viral particles that are better able to deliver their SHGP gene sequence payload into the cell (e.g. because there are more of them or because the SHGP enhances viral production) gain a selective advantage and can increase their representation in the population. This increases the number of cells that will generate viral particles harboring SHGP gene sequences that enhance viral production. In other words: Synthetic Helper Gene Products that enhance viral production enhance the packaging of their own coding DNA into more or better viral particles.

[00826] Example 10. Synthetic Helper Gene Product may be encoded in the genome of an RNA virus. [00827] In some embodiments, the Synthetic Helper Gene Product that is expressed by the virus may target RNA structures of the virus or the host.

[00828] In some embodiments, the Synthetic Helper Gene Product may disrupt protein- nucleic acid interactions. We show Synthetic Helper Gene Products can substantially increase viral production because of what is known in the art with respect to interactions between polypeptides and nucleic acids across all of biology.

[00829] Example 11. Antibody -like Synthetic Helper Gene Products Developed From Alternative Single Domain Antibody Scaffolds.

[00830] While we provide figures, data, and designs for antibody-like Synthetic Helper Gene Products or Proteins (SHGPs) based on a particular single domain antibody scaffolds (SEQ ID NOs: 215-216), it should be obvious to those skilled in the art that alternative single domain antibody scaffolds might be employed to develop alternative SHGPs.

[00831] The first step in developing alternative single domain antibody SHGPs based on alternative single domain antibody scaffolds would be to identify one or more alternative nanobody scaffolds. This is a trivial step for those skilled in the art, as there are databases that hold millions of different nanobody sequences. One such database is the Observed Antibody Sequence Database, which comprises antibody sequences from a variety of organisms, including humans and camels. A link to this database is active as of August 6th, 2024: <opig. stats .ox ac.uk/webapp8/oas/>. To find new nanobody scaffolds, which are exemplary single domain antibody scaffolds comprising the VHH domain of the camels (though other organisms like sharks are often used as well), one would simply search for camel antibody sequences. One may easily search for unpaired or paired camel antibody sequences and readily identify millions of nanobody scaffolds (i.e. VHH domains) that can be used as starting points for novel SHGPs.

[00832] While the scaffolds themselves do not have any inherent benefit as SHGPs and serve only as starting points, the critical aspect is how they are engineered to develop functional SHGPs. This process requires mutagenizing the antibody-like protein's residues that determine binding interactions. Those skilled in the art recognize that single antibodies, including single domain antibodies such as nanobodies, have distinct Complementarity Determining Regions (CDRs); nanobodies have 3 such CDRs. These CDRs are clearly marked in the Observable Antibody Database. The Observed Antibody Sequence database provides extensive sequence annotations and explicitly identifies the CDRs of single domain antibodies from camel antibodies in every case we have inspected. In the rare instance where a CDR might not be annotated, the sequence could either be skipped (the most practical option given the vast number of annotated sequences) or the CDR could be determined through a simple alignment and visual inspection. A simple sequence alignment (e.g., from UniProt) can be used to identify the putative CDRs one, two, and three. Those skilled in the art will recognize that each step in this process is trivial and straightforward.

[00833] Other databases might also be used to identify novel starting points for antibody-like SHGPs. Sticking with the nanobody example, one might go to UniProt and search for heavy domain (VHH) camelid antibodies (or other antibodies). Once the CDRs are identified, they can be easily mutagenized with saturation mutagenesis. Preferably, saturation mutagenesis targets all three CDRs; however, the sequence size becomes very large and hard to cover, though we have found that complete coverage of the sequence diversity is not necessary. If one wishes to limit sequence diversity to achieve a library size that is easier to cover, then one may target a single CDR, as we have taught in Example 1 and Fig. 14 (with expected results illustrated in Figs. 16-19 and 25-28).

[00834] Now that a starting nanobody scaffold and its critical sequence elements have been identified, the library fabrication physical steps can proceed. A DNA sequence can be synthesized using a standard DNA synthesis service provider (e.g. Integrated DNA Technologies, Twist Bioscience, Thermo Fisher Scientific). This DNA sequence should encode the amino acid sequence of the nanobody. This DNA sequence may optionally include degeneracies inside the CDRs. Alternatively, one may insert the DNA sequence into a cloning vector and generate the mutations in the CDRs using subsequent steps of saturation mutagenesis, for example, with eiPCR (enzymatic inverse PCR) with primers that have degeneracies on the 5' end and match the annealing region of the scaffold next to the CDR on the 3' end.

[00835] The benefit of the first approach with pre-installed degeneracies is that it is faster, easier, and simpler. The benefit of the second approach with a two-step DNA cloning strategy is that a variety of different mutagenesis strategies can be more easily employed and explored, for example, mutagenizing just CDR3 or mutagenizing CDR3 and another CDR. Another cloning strategy, as illustrated in Figure 14a, is to synthesize the nanobody with multiple cloning sites at the CDR3, allowing for degeneracies to be inserted via simple restriction-ligation cloning. This approach has the benefit of allowing the nanobody scaffold sequence to be directly cloned between AAV ITRs (SEQ ID NO: 8-9), which can sometimes be obstacles to downstream cloning and DNA manipulations involving PCR due to their complex DNA structures. It should be obvious to those skilled in the art that there are many potential cloning strategies one might employ to create a nanobody library with mutagenized CDRs, that there are many sequences that might serve as scaffolds for such SHGPs, and that it is obvious how CDRs can be identified and subsequently mutagenized to create SHG libraries. From this point, a new SHGP library has been created and can be subjected to selection as described in previous examples and as illustrated in other figures (e.g., Figs. 1-3).

[00836] SEQ ID NOs: 215-216 represent just a couple of the possible nanobody scaffold starting points, and it is obvious to those skilled in the art that alternative nanobodies could be used as starting points as described in this example. There are over 1 million different unique sequences in the Observed Antibody Sequence Database. A brief analysis of these sequences shows that there are many sequences that have little homology to one another (for example, under 60% homology), but they can still be used as nanobody scaffolds for SHGPs. SHGPs generated from alternative nanobody scaffolds through mutagenesis and the methods comprised here would be part of this invention.

[00837] Once candidate SHGPs from saturation mutagenesis show promising performance, they can be further optimized through directed evolution techniques if desired. One effective approach is to employ error-prone PCR (epPCR) to introduce random mutations throughout the entire gene sequence (as described in this specification for mutagenesis of engineered transcription factor SHGs). This method is particularly valuable for enhancing backbone or scaffold properties of antibody-like SHGPs, as it is often challenging to predict which residues outside the CDRs might improve activity. epPCR or other random mutagenesis techniques (e.g. chemical, UC, X-ray, shuffling of multiple hits) are preferred in this context because it allows for the exploration of a broader mutational space, potentially uncovering beneficial changes in unexpected regions of the protein’s scaffold (i.e. outside of the initially mutagenized CDRs). This approach can lead to improvements in protein stability, expression levels, or even subtle alterations in binding kinetics that were not targeted in the initial CDR- focused mutagenesis.

[00838] Instead of epPCR, saturation mutagenesis can be employed to fine-tune the binding and affinity profile of the SHGP. This approach might target CDRs that were not modified in the initial screening, or revisit previously mutated CDRs with a more focused library based on the results of the first round. By iteratively applying saturation mutagenesis to different CDRs or combinations of CDRs, it becomes possible to modulate the binding characteristics with high precision. This strategy allows for the optimization of not only binding affinity but also specificity, off-target interactions, and other critical parameters. [00839] A key aspect of this invention is the large-scale sequence-function mapping of SHGP variant sequences and their impact on viral titer. This approach is particularly amenable to machine-guided exploration of sequence space. The extensive data generated can be used to train Al models that can predict the effects of specific SHGP sequences. In this way, rationally designed saturation mutagenesis libraries may be developed using Al-guided prediction strategies. For instance, a particular degeneracy may encode a set of amino acids that are predicted to enhance the SHGP's performance. This method leverages the vast sequencefunction datasets already generated by techniques described in this invention, potentially accelerating the optimization process and improving outcomes.

[00840] Finally, the combination of broad exploration through epPCR and focused optimization via targeted saturation mutagenesis provides a powerful toolkit for refining SHGP candidates and maximizing their potential for the intended application. Employing additional techniques like Al-guided optimization can further enhance this process. These hybrid approaches allow researchers to benefit from the strengths of each method: the broad, unbiased exploration of epPCR; the precise, targeted modifications of saturation mutagenesis; and the data-driven insights of Al-guided design. By integrating these complementary strategies, it becomes possible to navigate the vast sequence space more efficiently, potentially uncovering novel SHGP variants with superior properties for specific applications.

[00841] Example 12. Antibody -like Synthetic Helper Gene Products Developed From Antibody Mimetic Scaffolds

[00842] While we provide figures, data, and designs for antibody-like Synthetic Helper Gene Products or Proteins (SHGPs) based on particular single domain antibody scaffolds (SEQ ID NOs: 215-216), it should be obvious to those skilled in the art that alternative antibody-like scaffolds might be employed to develop alternative SHGPs. One such alternative scaffold is the single-chain variable fragment (scFv), which consists of the variable regions of the heavy (VH) and light (VL) chains of an antibody connected by a flexible linker.

[00843] The first step in developing scFv-based SHGPs would be to identify one or more suitable scFv scaffolds. This is a straightforward process for those skilled in the art, as there are databases that hold numerous different antibody sequences. One such database is the Observed Antibody Sequence Database, which comprises antibody sequences from a variety of organisms, including humans, mice and other organisms. A link to this database is active as of August 6th, 2024: <opig. stats. ox. ac.uk/webapps/oas/>. To find scFv scaffolds, one would simply search for antibody sequences for an organism with the appropriate antibody structure (e.g. human with both heavy and light chain variable regions).

[00844] Once a suitable scaffold is identified, the next step is to design the scFv structure. This process involves selecting the VH and VL domains and connecting them with a suitable linker. The Observed Antibody Sequence Database provides extensive sequence annotations allowing easy identification of the relevant sequence elements allowing easy identification of the variable heavy and variable light chains (VH, VL, respectively). These will form the basis of the scFv structure: VH-linker-VL, where “linker” refers to a flexible linker sequence (for example GGGGSGGGGSGGGGS) which is used to connect the VH and VL domains. While a Gly-Ser linker is provided as an example, there are a variety of strategies covered in numerous publications for engineering optimal linkers that are known to those skilled in the art.

[00845] With the scFv structure in place, the next step is to develop mutagenesis strategies for optimization. As described in Example 11 for nanobodies, the scFv scaffold is useless on its own and must be engineered to develop functional SHGPs that are capable of improving viral production. This process requires mutagenizing the antibody-like protein's residues that determine binding interactions, which in the case of scFvs are the six CDRs (three in VH and three in VL) followed by functional evaluation (e.g. selections or screens). The Observed Antibody Sequence Database provides extensive sequence annotations allowing easy identification of the relevant sequence elements (exemplary fields include "cdrl aa heavy", "cdr2_aa_heavy", "cdr3_aa_heavy", "cdrl_aa_light", "cdr2_aa_light", "cdr3_aa_light" and should carry obvious meaning to those skilled in the art). The scFv’s CDR sequences and their positions within the scaffold sequence correspond to the portion of the sequence that are most preferably mutagenized with saturation mutagenesis in order to create a SHGP library.

[00846] To design the mutagenesis strategy for the CDRs, replace each amino acid in one or more of the six CDR regions with X (which corresponds to a degenerate amino acid, which can be created using a degenerate codon such as NNK). This represents a complete randomization of these regions. Preferably target one or more CDR sequences for mutagenesis and maintain the surrounding framework regions intact. An example scFv mutagenesis design with brackets [XXX. . X ] denoting the CDR regions in the scFv sequence would look like the following:

[00847] VH-FR1-[XXXXX]-VH-FR2-[XXXXXX]-VH-FR3-[XXXXXXXXX]-VH- FR4-linker-VL-FRl-[XXXXX]-VL-FR2-[XXX]-VL-FR3-[XXXXXXX]-VL-FR4. [00848] This sequence represents a mutagenized scFv design, ready for library creation. VH represents the variable heavy chain domain; VL represents the variable light chain domain; FR1, FR2, FR3, and FR4 represent the framework regions in each domain; [XXXXX] represents the mutagenized CDR regions (CDR1, CDR2, and CDR3 in each domain); "linker" represents the flexible peptide sequence connecting VH and VL. [00849] When creating the library, it's important to balance diversity with manageable library sizes. While complete randomization of all six CDRs provides the greatest diversity, it may result in library sizes that are challenging to screen comprehensively. Strategies such as focusing on specific CDRs or using smart library design techniques can help in covering the sequence space effectively. Similar to what is described in Example 11, if one wishes to limit sequence diversity to achieve a library size that is easier to cover, then one may target a single CDR or use degenerate codons that constrain diversity.

[00850] For library creation and cloning, several strategies can be employed, similar to those described in Example 11. A DNA sequence can be synthesized using a standard DNA synthesis service provider. This DNA sequence should encode the amino acid sequence of the scFv scaffold designed as described above or with a comparable approach. This DNA sequence may optionally include degeneracies inside the CDRs. It is obvious that with the additional CDRs (6 instead of a nanobody's 3), the library fabrication is more complex than for nanobody SHGPs. However, with continued developments in DNA synthesis technology, the economics and ease with which high complexity DNA libraries can be synthesized will improve, making scFv SHGPs easier to develop. Alternatively, one may insert the scFv DNA sequence into a cloning vector and generate the mutations in the CDRs using subsequent steps of saturation mutagenesis as described in Example 11. The same considerations enumerated in Example 11 also apply to scFvs.

[00851] Once the SHG DNA library has been created, it can be subjected to selection as described in previous examples and as illustrated in other figures (e.g., Figs. 1-3). Once SHGPs are identified, they can optionally be further optimized through directed evolution techniques as described in Example 11.

[00852] In conclusion, it should be obvious to those skilled in the art that there are many equivalent antibody-like proteins that might serve as scaffolds for developing SHGPs. This example teaches an equivalent approach for the use of scFvs as scaffolds for developing SHGPs.

[00853] Example 13. SHGPs Based on Alternative Engineered Transcription Factors Scaffolds

[00854] While previous examples have focused on antibody-like scaffolds, this example demonstrates how transcription factors can be engineered to create effective SHGPs. The unique properties of transcription factors, including their ability to bind DNA and influence gene expression, make them particularly well-suited for this application. [00855] The first step in developing engineered Transcription Factor-based synthetic helper gene proteins is to identify novel scaffolds. This can be easily accomplished by searching protein sequence databases, preferably the UniProt database.

[00856] As of August 6th, 2024, a simple search for "transcription factor" in the UniProt database yields over 9.8 million results. To refine this sequence space, one can use an advanced search with specific parameters. By setting the search field to "All", using "transcription factor" as the search term, limiting the sequence length to between 30 and 300 amino acids, and applying a taxonomy filter to exclude Homo sapiens, the search can be narrowed down significantly. This refined approach helps in identifying potential transcription factor scaffolds that are more suitable for SHGP development.

[00857] This example search yields over 4 million different transcription factors from diverse sources. It is important to note that this is just one possible search strategy; many other potential searches could be performed.

[00858] Given the abundance of options, it is evident to one skilled in the art that choosing novel transcription factors as scaffold starting points is a straightforward process. The transcription factors specified in our sequence listing (SEQ ID NOs: 217-221) should be considered non-limiting examples, as it is clear that similar results can be achieved using different transcription factors.

[00859] A multiple sequence alignment performed on SEQ ID NOs: 217-221 using UniProt's alignment tool reveals that the percent sequence identity between and across all five sequences ranges from 23% to 35%, including the added nuclear localization sequence tag. When this tag is removed, the percent identity drops to between 11% and 28%.

[00860] This analysis demonstrates that there are few sequence elements specific to any given transcription factor, and that virtually any type of transcription factor can serve as a good synthetic helper gene scaffold. This rationale guided our selection of the five exemplary synthetic helper genes described in this patent specification, which should be considered nonlimiting examples.

[00861] From this large set of 4 million sequences, some subsets can be chosen. While the choice is somewhat arbitrary, we recommend selecting transcription factors that have somewhat globular domains. We believe that microbial transcription factors are preferred, as demonstrated by the sequences listed in the specification. While not all sequences will have crystal structures, new capabilities like AlphaFold can provide structure predictions. A good rule of thumb is to choose diverse sequences with varied structures. [00862] When selecting transcription factor scaffolds, preferred embodiments will include motifs classically associated with transcription factors. These may include the Helix- Tum-Helix (HTH), characterized by two a-helices connected by a short sequence, with the second helix involved in direct DNA interaction; the Zinc Finger, comprising a zinc ion coordinated by cysteine and/or histidine residues, forming a structure that binds to the major groove of DNA; the Leucine Zipper (bZIP), which includes a basic region that interacts with DNA and a leucine zipper for dimerization, essential for binding specificity and stability; the Helix-Loop-Helix (HLH), consisting of two a-helices separated by a loop, with functions in both DNA binding and protein dimerization; and the Homeodomain, featuring three a-helices, particularly the recognition helix, which is crucial for specific DNA interactions. These should be considered non-limiting examples, as other motifs may also be suitable for SHGP development.

[00863] When developing new transcription factors for SHGs, if the protein sequence includes a nuclear localization sequence (NLS) that is expected to be compatible with a human system, such as those from mouse or primate transcription factors, no additional NLS needs to be added. However, when using a transcription factor derived from a microbial system, as described in this specification, an NLS should be added along with other sequence elements required for proper expression. Other domains may optionally be added (activation domains, inhibition domains, methylation domains, etc. . .). Protein sequences can be easily reverse translated using tools like IDT DNA's Reverse Translate tool or a custom script (e.g. in python), allowing codon optimization for a human host. This optimized DNA can then be ordered, synthesized, and delivered. The DNA encoding the synthetic helper gene should also comprise other elements required for expression, such as a promoter, Kozak sequence, poly-A tail, T2A, and terminator (if applicable).

[00864] To develop the transcription factor scaffold into an engineered transcription factor synthetic helper gene, mutagenesis and selection or screening must be performed. A variety of mutagenesis approaches can be employed, ranging from targeted and saturation mutagenesis to random mutagenesis, such as error-prone PCR, chemical mutagenesis, or radiation mutagenesis. In this patent, we exemplify the development of engineered transcription factors based SHGPs using error-prone PCR-based approaches due to the method’s simplicity, speed, and cost-effectiveness. However, other methods like saturation mutagenesis targeting DNA binding residues could also be used, in an approach conceptually similar to targeting the CDR of antibody-like synthetic helper genes. [00865] The choice of mutagenesis strategy can significantly impact the diversity and functionality of the resulting SHGP library. While error-prone PCR offers a straightforward approach to generating random mutations throughout the sequence, targeted mutagenesis of DNA-binding regions can allow for more focused exploration of sequence space. The optimal strategy may depend on factors such as the specific transcription factor scaffold, the desired library size, and the available screening or selection methods.

[00866] The mutagenized synthetic helper gene library based on transcription factors should be cloned into a viral transfer vector capable of being packaged into a viral particle. Figure 15 exemplifies an AAV architecture where the mutagenized transcription factor-based helper gene library is inserted into a transfer vector and flanked by AAV ITR sequences. This DNA sequence, if transfected into a cell configured to produce AAV would be expected to be packaged into a viral particle.

[00867] Once the library has been created and cloned into the appropriate vector, the selection process can begin. This crucial step allows for the identification of SHGPs that effectively enhance viral production.

[00868] At this point, the library is treated like any other synthetic helper gene library and can be subjected to enrichment (as described in Figs. 1-3 ). Briefly, the library is transfected into viral-producing cells where the ITR-flanked synthetic helper gene library variants begin to be packaged into viral particles. Simultaneously, the SHG is expressed, generating variant transcription factor SHGPs. These proteins, with NLSs, then localize to the nucleus and, depending on their DNA binding profiles resulting from mutagenesis, may bind to various genomic DNA sequences. It is important to note that the transcription Factor does not need to have a defined DNA binding profile, we are essentially starting with a protein that should be capable of binding DNA and then substantially mutagenizing at such that it has some new and unknown DNA binding profile, this may be binding to one DNA sequence with high specificity and Affinity or it may be a much more promiscuous DNA binding profile, this is less important but what is more important is the biological perturbation that results.

[00869] This transcription factor binding to genomic DNA will cause perturbations to the cell as a result of changes in DNA expression, resulting from a range of different mechanisms such as altering DNA packing tightness, roadblock interference, and enhancing or disrupting molecular associations that determine gene expression. Cumulatively, these changes can be described as a perturbation resulting from the SHGP. If this SHGP-induced cellular perturbation improves the cell's ability to produce viral particles, then the efficiency with which the synthetic helper gene DNA is packaged will be enhanced. Conversely, if the perturbation reduces the cell's ability to generate viral particles, the synthetic helper gene sequence will be less efficiently packaged and may ultimately be depleted or eliminated from the library. Thus, the SHGP influences its ability to package its own SHG coding nucleotide sequence.

[00870] This process couples the physiological impact of each Synthetic Helper Gene Product on viral packaging to the Synthetic Helper Gene sequence's ability to propagate, establishing a strong selective pressure for Synthetic Helper Genes that enhance viral titer or function/infectivity. Representative examples can be found in Figs. 29-33 Part B, which show histograms of SHG library enrichment during such a selection. These histograms illustrate how engineered transcription factor SHGPs that increase viral production will increase their representation in the library as a result of reprogramming cells to become better viral production factories are enriched (shown as bars with positive log scores), whereas transcription factors that reduce viral production are depleted (shown as bars with enrichment factors less than 0). These representative results are expected for synthetic helper genes derived from any other transcription factor scaffold starting points that can be selected as described above, for example, from UniProt searches.

[00871] After initial selection, further optimization of promising SHGP candidates can be achieved through iterative rounds of mutagenesis and selection as described in examples 11 and 12. This process allows for fine-tuning of the SHGPs' ability to enhance viral production. Additionally, the extensive sequence-function data generated during this process can be leveraged for Al-guided optimization, potentially accelerating the development of highly effective SHGPs.

[00872] In conclusion, engineered transcription factor-based SHGPs offer a powerful and flexible approach to enhancing viral production that can be developed from a wide variety of different transcription factor scaffolds and the invention should not be considered to be limited to SEQ ID NOs: 217-221. The ability to select from a vast array of transcription factor scaffolds, combined with targeted mutagenesis and selection strategies, provides numerous opportunities for developing effective SHGPs.

[00873] Example 14. Identifying Motifs Present in SHGPs

[00874] The identification of motifs can provide valuable insights into the structurefunction relationships of SHGPs and guide further optimization efforts and can serve to delineate sequences that are included or excluded from this invention.

[00875] The first step in identifying motifs is to categorize amino acid residues based on their side chain properties. For this purpose, we group the 20 standard amino acids into five categories: hydrophobic (HYD: 'G', 'A', 'V, T, 'L', 'M', 'P'), aromatic (ARO: 'F', 'W, 'Y'), polar uncharged (POL: 'S', 'T', 'Q', 'N', 'C'), positively charged (POS: 'K', 'R', 'H'), and negatively charged (NEG: 'D', 'E'). This categorization allows for a simplified representation of the SHGP sequence while still capturing important physicochemical properties.

[00876] To identify motifs, we use a sliding window approach. A window of a fixed length (preferably about 5 amino acids) is moved along the SHGP sequence one residue at a time. For each window position, the amino acid sequence is converted into a string of category codes based on the groupings described above. For example, the sequence "GSTKA" would be converted to "HYD POL POL POS HYD". Each unique motif encountered during this process is recorded.

[00877] This approach allows for the systematic identification of recurring patterns within SHGPs. For instance, we might find that successful SHGPs often contain motifs such as "HYD HYD POL ARO NEG" or "POS POL HYD HYD HYD". These motifs can provide insights into the structural elements that contribute to SHGP function.

[00878] This motif identification approach can be applied to compare SHGPs derived from different scaffolds, such as the antibody-like and transcription factor-based SHGPs described in previous examples. By identifying common motifs across these different SHGP types, we may uncover fundamental sequence patterns that contribute to enhanced viral production, regardless of the scaffold origin. For example, we find that all 20 motifs have at least two or more HYDs, suggesting the importance of hydrophobicity. This informs decision making in selecting scaffolds (e.g. as described in Examples 11-13), where proteins that have fewer hydrophobic residues (e.g. potentially more intrinsically disordered) might be passed over in favor of scaffolds possessing one or more of the motifs listed in the Synthetic Helper Motifs table (selected motifs of length 5 are indicated):

[00879] HYD HYD HYD POL HYD,

[00880] HYD POL HYD HYD HYD, [00881] HYD HYD HYD HYD HYD,

[00882] HYD HYD POL HYD HYD,

[00883] HYD HYD HYD HYD POL,

[00884] POL HYD HYD HYD HYD,

[00885] POL HYD HYD HYD POL,

[00886] HYD HYD POS HYD HYD,

[00887] HYD HYD HYD POS HYD,

[00888] HYD POL POL HYD HYD, [00889] POL HYD HYD POL HYD,

[00890] HYD HYD POL HYD POL,

[00891] HYD HYD NEG POL HYD,

[00892] HYD HYD NEG HYD HYD,

[00893] HYD NEG POL HYD HYD, [00894] POS POL HYD HYD HYD,

[00895] POL HYD POL HYD HYD,

[00896] NEG HYD HYD HYD HYD, [00897] HYD POL POL HYD POL,

[00898] POL HYD POL POL HYD.

[00899] In conclusion, the identification and analysis of motifs in SHGPs provide a useful tool for understanding and engineering the sequence-function relationships of SHGPs. This approach not only aids in the characterization of existing SHGPs but also guides the design and optimization of new ones. By focusing on scaffolds that possess the motifs identified as beneficial for SHGP function, we may streamline the SHGP development and engineering process.

[00900] Other Embodiments.

[00901] The detailed description set forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.

[00902] References Cited

[00903] All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

[00904]

Claims

1. A method of obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of a viral vector composition, the method comprising:

(a) culturing a first plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a Synthetic Helper Gene Product encoded by a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the first nucleotide sequence within the viral capsids or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to the one or more viral-specific packaging sequences necessary for encapsulation of the second nucleotide sequence within the viral capsids, thereby obtaining a first plurality of viral vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps (bl)-(b2) one or more times in cycles:

(bl) allowing a plurality of viral vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for replication of the virus; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences producing the Synthetic Helper Gene Product, thereby obtaining a plurality of viral vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of viral vectors or the plurality of viral vectors of the present cycle to infect a final plurality of host cells; and

(d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of the viral vector composition by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from (i) the final plurality of host cells and/or (ii) a final plurality of viral vectors produced in the final plurality of host cells.

2. The method of claim 1, wherein in step (a), the first nucleotide sequence is operably linked to the one or more viral-specific packaging sequences, and the method comprises culturing the final plurality of host cells under conditions suitable for recombinant viral production, wherein each host cell of the final plurality of host cells comprises the elements

(i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to one or more viral-specific packaging sequences and producing the Synthetic Helper Gene Product encoded by the first nucleotide sequence, thereby producing the final plurality of viral vectors from the final plurality of host cells and determining the Synthetic Helper Gene Product by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from the final plurality of viral vectors.

3. The method of claim 1, wherein

(i) the viral vector composition is an adeno-associated virus (AAV) vector composition;

(ii) the at least one viral replication gene comprises at least one AAV replication gene;

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene;

(iv) the at least one additional viral gene comprises at least one AAV helper gene; and

(v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs).

4. The method of claim 1, wherein

(i) the viral vector composition is a lentivirus vector composition;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene;

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene and at least one env gene;

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene; and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence.

5. The method of claim 1, wherein each host cell of first plurality of host cells and each host cell of final plurality of host cells are mammalian host cells.

6. The method of claim 1, wherein the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences further encodes a reporter, a therapeutic payload or a selectable marker.

7. The method of claim 1, wherein the first plurality of host cells at step (a) comprises at least 1,000 host cells each producing a unique, structurally different Synthetic Helper Gene Product.

8. The method of claim 1, further comprising (e): generating new viral vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the viral vector composition of increased viral titer and/or transduction efficiency.

9. The method of claim 8, which produces the viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from the group consisting of viral titer and transduction efficiency.

10. The method of claim 8, wherein the Synthetic Helper Gene Product is not essentially present in the viral vector composition of increased viral titer and/or transduction efficiency.

11. The method of claim 8, wherein generating new viral vectors comprises: culturing a new plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the new plurality of host cells comprises the Synthetic Helper Gene Product determined in (d) and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload.

12. A plurality of host cells permissive for replication of a virus, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and (iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; wherein the Synthetic Helper Gene Product increases a characteristic of viral vectors produced by the plurality of host cells by at least 2-fold compared to a corresponding characteristic of viral vectors produced by a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of viral vectors is selected from the group consisting of viral titer and transduction efficiency.

13. The plurality of host cells of claim 12, wherein

(i) the virus is an adeno-associated virus (AAV);

(ii) the at least one viral replication gene comprises at least one AAV replication gene;

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene;

(iv) the at least one additional viral gene comprises at least one AAV helper gene; and

(v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs).

14. The plurality of host cells of claim 12, wherein

(i) the virus is a lentivirus;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene;

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene and at least one env gene;

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene; and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence.

15. The plurality of host cells of claim 12, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

16. The plurality of host cells of claim 12, wherein each host cell of the plurality of host cells is a mammalian host cell.

17. The plurality of host cells of claim 12, wherein each host cell of the plurality of host cells is an insect host cell.

18. The plurality of host cells of claim 12, wherein the payload comprises a therapeutic gene.

19. The plurality of host cells of claim 12, wherein the plurality of host cells comprises at least 10,000 host cells.

20. The plurality of host cells of claim 12, wherein the Synthetic Helper Gene Product comprises an amino acid sequence having at least 90% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 35 - SEQ ID NO: 221.

21. The plurality of host cells of claim 12, wherein the Synthetic Helper Gene Product comprises an amino acid sequence selected from the group consisting of: HYD-HYD- HYD-POL-HYD, HYD-POL-HYD-HYD-HYD, HYD-HYD-HYD-HYD-HYD, HYD- HYD-POL-HYD-HYD, HYD-HYD-HYD-HYD-POL, POL-HYD-HYD-HYD-HYD, POL-HYD-HYD-HYD-POL, HYD-HYD-POS-HYD-HYD, HYD-HYD-HYD-POS-HYD, HYD-POL-POL-HYD-HYD, POL-HYD-HYD-POL-HYD, HYD-HYD-POL-HYD-POL, HYD-HYD-NEG-POL-HYD, HYD-HYD-NEG-HYD-HYD, HYD-NEG-POL-HYD- HYD, POS-POL-HYD-HYD-HYD, POL-HYD-POL-HYD-HYD, NEG-HYD-HYD-HYD- HYD, HYD-POL-POL-HYD-POL, and POL-HYD-POL-POL-HYD, wherein HYD is one of the following amino acid residues: 'G', 'A', 'V, T, 'L', 'M', 'P'; ARO is one of the following amino acid residues: 'F', 'W, 'Y'; POL is one of the following amino acid residues: 'S', 'T', 'Q', 'N', 'C; POS is one of the following amino acid residues: 'K', 'R', H'; and NEG is one of the following amino acid residues: 'D', 'E.

22. A method of producing a viral vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(a) culturing a plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; and

(b) producing the viral vector composition of increased viral titer and/or transduction efficiency from the plurality of host cells, wherein the viral vector composition has an increased viral titer and/or transduction efficiency which is at least a 20% greater than a viral titer and/or transduction efficiency of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product.

23. The method of claim 22, wherein

(i) the viral vector composition is an adeno-associated virus (AAV) vector composition;

(ii) the at least one viral replication gene comprises at least one AAV replication gene;

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene;

(iv) the at least one additional viral gene comprises at least one AAV helper gene; and

(v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs).

24. The method of claim 22, wherein

(i) the viral vector composition is a lentivirus vector composition;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene;

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene and at least one env gene;

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene; and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence.

25. The method of claim 22, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

26. The method of claim 22, wherein the Synthetic Helper Gene Product is exogenously supplied to each host cell of the plurality of host cells.

27. The method of claim 22, wherein each host cell of the plurality of host cells is a mammalian host cell.

28. The method of claim 22, wherein each host cell of the plurality of host cells is an insect host cell.

29. The method of claim 22, wherein the plurality of host cells comprises at least 10,000 host cells.

30. The method of claim 22, wherein the Synthetic Helper Gene Product is not essentially present in the viral vector composition of increased viral titer and/or transduction efficiency.

31. The method of claim 22, wherein the Synthetic Helper Gene Product comprises an amino acid sequence having at least 90% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 35 - SEQ ID NO: 221.

32. The method of claim 22, wherein the Synthetic Helper Gene Product comprises an amino acid sequence selected from the group consisting of: HYD-HYD-HYD-POL-HYD, HYD-POL-HYD-HYD-HYD, HYD-HYD-HYD-HYD-HYD, HYD-HYD-POL-HYD- HYD, HYD-HYD-HYD-HYD-POL, POL-HYD-HYD-HYD-HYD, POL-HYD-HYD- HYD-POL, HYD-HYD-POS-HYD-HYD, HYD-HYD-HYD-POS-HYD, HYD-POL-POL- HYD-HYD, POL-HYD-HYD-POL-HYD, HYD-HYD-POL-HYD-POL, HYD-HYD- NEG-POL-HYD, HYD-HYD-NEG-HYD-HYD, HYD-NEG-POL-HYD-HYD, POS-POL- HYD-HYD-HYD, POL-HYD-POL-HYD-HYD, NEG-HYD-HYD-HYD-HYD, HYD- POL-POL-HYD-POL, and POL-HYD-POL-POL-HYD, wherein HYD is one of the following amino acid residues: 'G', 'A', 'V, T, 'L', 'M', 'P'; ARO is one of the following amino acid residues: 'F', 'W, 'Y'; POL is one of the following amino acid residues: 'S', 'T', 'Q', 'N', 'C; POS is one of the following amino acid residues: 'K', 'R', 'H'; and NEG is one of the following amino acid residues: 'D', E'.

33. A plurality of host cells permissive for replication of a virus, wherein each host cell of the plurality of host cells comprises a Synthetic Helper Gene Product and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload; wherein the plurality of host cells comprises at least 100,000 host cells each comprising structurally different Synthetic Helper Gene Products.

34. The plurality of host cells of claim 33, wherein the Synthetic Helper Gene Product is produced ribosomally in each host cell of the plurality of host cells.

35. The plurality of host cells of claim 34, wherein at least one hundred of structurally different Synthetic Helper Gene Products produced in the at least 1,000,000 host cells each increases a characteristic of viral vectors produced by the host cell by at least 2-fold compared to a corresponding characteristic of viral vectors produced by a reference host cell under essentially identical conditions, wherein the reference host cell comprises the elements (i)-(iv) of the plurality of host cells and does not comprise the Synthetic Helper Gene Product, and wherein the characteristic of viral vectors is selected from the group consisting of viral titer and transduction efficiency.

36. The plurality of host cells of claim 33, wherein

(i) the virus is an adeno-associated virus (AAV);

(ii) the at least one viral replication gene comprises at least one AAV replication gene;

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene;

(iv) the at least one additional viral gene comprises at least one AAV helper gene; and

(v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs).

37. The plurality of host cells of claim 33, wherein

(i) the virus is a lentivirus;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene;

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene and at least one env gene;

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene; and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence.

38. The plurality of host cells of claim 33, wherein each host cell of plurality of host cells are mammalian host cells.

39. The plurality of host cells of claim 33, wherein the Synthetic Helper Gene Product comprises an amino acid sequence having at least 90% or more sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 35 - SEQ ID NO: 221.

40. The plurality of host cells of claim 33, wherein the Synthetic Helper Gene Product comprises an amino acid sequence selected from the group consisting of: HYD-HYD- HYD-POL-HYD, HYD-POL-HYD-HYD-HYD, HYD-HYD-HYD-HYD-HYD, HYD- HYD-POL-HYD-HYD, HYD-HYD-HYD-HYD-POL, POL-HYD-HYD-HYD-HYD, POL-HYD-HYD-HYD-POL, HYD-HYD-POS-HYD-HYD, HYD-HYD-HYD-POS-HYD, HYD-POL-POL-HYD-HYD, POL-HYD-HYD-POL-HYD, HYD-HYD-POL-HYD-POL, HYD-HYD-NEG-POL-HYD, HYD-HYD-NEG-HYD-HYD, HYD-NEG-POL-HYD- HYD, POS-POL-HYD-HYD-HYD, POL-HYD-POL-HYD-HYD, NEG-HYD-HYD-HYD- HYD, HYD-POL-POL-HYD-POL, and POL-HYD-POL-POL-HYD, wherein HYD is one of the following amino acid residues: 'G', 'A', 'V, T, 'L', 'M', 'P'; ARO is one of the following amino acid residues: 'F', 'W, 'Y'; POL is one of the following amino acid residues: 'S', 'T', 'Q', 'N', 'C; POS is one of the following amino acid residues: 'K', 'R', H'; and NEG is one of the following amino acid residues: 'D', 'E.

41. A method of producing a viral vector composition of increased viral titer and/or transduction efficiency, the method comprising:

(A) obtaining a Synthetic Helper Gene Product capable of increasing viral titer and/or transduction efficiency of a viral vector composition by performing the following steps:

(a) culturing a first plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the first plurality of host cells comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a Synthetic Helper Gene Product encoded by a first nucleotide sequence, wherein (v) the first nucleotide sequence is operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the first nucleotide sequence within the viral capsids or (vi) the first nucleotide sequence is associated with a second nucleotide sequence comprising a barcode that comprises identifying information regarding the Synthetic Helper Gene Product produced in the host cell, and the second nucleotide sequence is operably linked to the one or more viral-specific packaging sequences necessary for encapsulation of the second nucleotide sequence within the viral capsids, thereby obtaining a first plurality of viral vectors comprising the first nucleotide sequence and/or the second nucleotide sequence from the first plurality of host cells;

(b) optionally, repeating the following steps (bl)-(b2) one or more times in cycles:

(bl) allowing a plurality of viral vectors of the previous cycle to infect a plurality of host cells of the present cycle permissive for replication of the virus; and

(b2) culturing the plurality of host cells of the present cycle under conditions suitable for recombinant viral production, wherein each host cell of the plurality of host cells of the present cycle comprises the elements (i)-(iii) of the first plurality of host cells, and further comprises the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences producing the Synthetic Helper Gene Product, thereby obtaining a plurality of viral vectors of the present cycle comprising the first nucleotide sequence;

(c) allowing the first plurality of viral vectors or the plurality of viral vectors of the present cycle to infect a final plurality of host cells; and

(d) determining one or more Synthetic Helper Gene Products capable of increasing viral titer and/or transduction efficiency of the viral vector composition by analyzing nucleotide sequences operably linked to the one or more viral-specific packaging sequences from (i) the final plurality of host cells and/or (ii) a final plurality of viral vectors produced in the final plurality of host cells; and

(B) generating new viral vectors in the presence of a Synthetic Helper Gene Product determined in step (d), thereby producing the viral vector composition of increased viral titer and/or transduction efficiency.

42. The method of claim 41, which produces the viral vector composition having a characteristic, which is at least 2-fold higher than a corresponding characteristic of a reference viral vector composition produced in a plurality of reference host cells under essentially identical conditions, wherein each reference host cell of the plurality of reference host cells comprises the elements (i)-(iii) of the first plurality of host cells and does not comprise the first nucleotide sequence and the Synthetic Helper Gene Product, and wherein the characteristic is selected from the group consisting of viral titer and transduction efficiency.

43. The method of claim 41, wherein the Synthetic Helper Gene Product is not essentially present in the viral vector composition of increased viral titer and/or transduction efficiency.

44. The method of claim 41, wherein the first plurality of host cells comprises at least 100,000 host cells each comprising structurally different Synthetic Helper Gene Products.

45. The method of claim 41, wherein

(i) the viral vector composition is an adeno-associated virus (AAV) vector composition;

(ii) the at least one viral replication gene comprises at least one AAV replication gene;

(iii) the at least one viral structural gene comprises at least one AAV capsid encoding gene;

(iv) the at least one additional viral gene comprises at least one AAV helper gene; and

(v) the one or more viral-specific packaging sequences comprise at least two functional AAV inverted terminal repeats (ITRs).

46. The method of claim 41, wherein

(i) the viral vector composition is a lentivirus vector composition;

(ii) the at least one viral replication gene comprises at least one lentiviral pol gene;

(iii) the at least one viral structural gene comprises at least one lentiviral gag gene and at least one env gene;

(iv) the at least one additional viral gene comprises at least one lentiviral rev gene; and

(v) the one or more viral-specific packaging sequences comprise a Psi sequence.

47. The method of claim 41, wherein each host cell of first plurality of host cells and each host cell of final plurality of host cells are mammalian host cells.

48. The method of claim 41, wherein the first nucleotide sequence operably linked to the one or more viral-specific packaging sequences further encodes a reporter, a therapeutic payload or a selectable marker.

49. The method of claim 41, wherein generating new viral vectors comprises: culturing a new plurality of host cells permissive for replication of a virus under conditions suitable for recombinant viral production, wherein each host cell of the new plurality of host cells comprises the Synthetic Helper Gene Product determined in (d) and further comprises:

(i) at least one viral replication gene essential for the replication of the virus;

(ii) at least one viral structural gene essential for formation of viral capsids of the virus;

(iii) at least one additional viral gene necessary to produce the virus in the host cells; and

(iv) a nucleotide sequence operably linked to one or more viral-specific packaging sequences necessary for encapsulation of the nucleotide sequence within the viral capsids, wherein the nucleotide sequence encodes a payload.