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  • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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  • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
  • C12N2740/15051—Methods of production or purification of viral material
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  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
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  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16051—Methods of production or purification of viral material
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14151—Methods of production or purification of viral material
  • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

• Viral vector-mediated gene transfer is a valuable tool for studying gene functions and gene therapeutics.
• production of viral vectors comprising certain transgenes e.g. , transgene products that are toxic to packaging cells or incompatible with viral vector packaging systems
• challenges for example, very low titer or no production of viral vectors.
• the disclosure relates to abolishing transgene expression by RNA degradation (e.g. , as mediated by short-hairpin RNAs (shRNAs), artificial miRNAs (amiRNAs), etc.) during the vector packaging process to allow for efficient production of vectors (e.g. , viral vectors) comprising transgene products that are cytotoxic and/or incompatible with a viral vector packaging system.
• RNA degradation e.g. , as mediated by short-hairpin RNAs (shRNAs), artificial miRNAs (amiRNAs), etc.
• shRNAs short-hairpin RNAs
• amiRNAs artificial miRNAs
• the disclosure provides a method for controlling or improving recombinant virus production yield comprising: introducing into a host cell a first nucleic acid comprising a transgene; introducing into the host cell a second nucleic acid capable of expressing an interfering nucleic acid, wherein the interfering nucleic acid specifically inhibits expression of the transgene; replicating the nucleic acid comprising the transgene within the host cell; and, optionally, isolating a virus particle comprising the first nucleic acid from the host cell.
• the first nucleic acid and the second nucleic acid are introduced into the host cell simultaneously. In some embodiments, the first nucleic acid and the second nucleic acid are introduced into the host cell separately. It should be appreciated that, in some embodiments, the first nucleic acid and the second nucleic acid are located on the same plasmid. In some embodiments, the inhibition of transgene expression by the second nucleic acid is transient. In some embodiments, the inhibition of transgene expression by the second nucleic acid is permanent. In some embodiments, a host cell is a viral vector packaging cell. In some embodiments, the host cell is a mammalian cell.
• a mammalian cell is a human cell, for example a HEK293T cell.
• a host cell is an insect cell.
• a host cells is an insect cell, for example a Spodoptera frugiperda (Sf9) cell.
• a first nucleic acid is a lentiviral transfer plasmid, an adeno- associated virus (AAV) vector, an adenovirus (Ad) vector, or a retroviral vector.
• a first nucleic acid is a lentiviral transfer plasmid and comprises at least one long terminal repeat (LTR).
• a first nucleic acid is an AAV vector and comprises at least one inverted terminal repeat (ITR).
• a first nucleic acid is a retroviral transfer plasmid and comprises at least one long terminal repeat (LTR).
• an AAV ITR is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 ITR.
• an AAV vector is a self-complementary AAV (scAAV) vector comprising at least one AITR or mutant ITR (mTR).
• expression of a transgene interferes with viral vector packaging (e.g. , via a product of the transgene) in a host cell.
• the transgene is cytotoxic or forms a secondary structure with high thermal stabilities (e.g. , has one or more physiochemical properties that are detrimental to the fitness of the host cell).
• a transcript encoded by the first nucleic acid comprises one or more binding sites for an inhibitory nucleic acid. In some embodiments, one or more binding sites are located between the last codon and the polyA tail of the transcript. In some
• one or more binding sites are located in a 5' untranslated region (5'UTR) of the transcript. In some embodiments, one or more binding sites are located between the last codon and the polyA tail of the transcript and one or more binding sites are located in a 5'UTR of the transcript. In some embodiments the one or more binding sites comprise a miR-333 binding site (SEQ ID NO: 3), a miR-865 binding site (SEQ ID NO: 4), or a combination thereof. In some embodiments, the one or more binding sites are 1, 2, or 3 binding sites.
• an inhibitory nucleic acid is a micro-RNA (miRNA) or an artificial miRNA (amiRNA).
• miRNA micro-RNA
• amiRNA artificial miRNA
• the sequence of an inhibitory nucleic acid binding site is not recognized by endogenous miRNAs of a host cell.
• a second nucleic acid expresses a short hairpin RNA, miRNA, or an amiRNA. In some embodiments, the second nucleic acid expresses a miRNA or an amiRNA. In some embodiments, a miRNA or amiRNA comprises a miRNA sequence expressed in a non- human cell (e.g. , a miRNA sequence that is naturally expressed only in non-human cells). In some embodiments, a miRNA or amiRNA comprises a miRNA sequence expressed in an insect cell or a plant cell (e.g. , a miRNA sequence that is naturally expressed only in an insect cell or a plant cell). In some embodiments, a miRNA or amiRNA comprises a miRNA sequence expressed in a plant cell (e.g.
• an miRNA or amiRNA comprises a miR-333 sequence (SEQ ID NO: 1) or a miR-856 sequence (SEQ ID NO: 2).
• an amiRNA comprises a miR-30 scaffold (e.g. , backbone sequence, such as a pri-miR30a backbone sequence).
• a transcript comprises one or more binding sites for a miR-333 sequence or a miR-856 sequence.
• a host cell further comprises one or more accessory plasmids.
• one or more accessory plasmids are a packaging plasmid, an Env encoding plasmid, a Rev encoding plasmid, a Rep encoding plasmid, or a Cap encoding plasmid.
• a transgene expressed from the virus particle isolated from the host cell is functional.
• FIG. 1 shows non-limiting examples of viral and non- viral vectors.
• vectors shown in FIG. 1 are useful for delivery of one or more transgenes to a subject (e.g. , a cell of a subject).
• FIGs. 2A-2C show palindrome sequences compromise rAAV genome homogeneity and yield.
• FIG. 2A shows a schematic of self-complementary AAV (scAAV) plasmids comprising a CMV enhancer/chicken ⁇ -actin promoter (CB), an EGFP reporter gene, and a beta-globin polyA sequence (PA).
• shRNA cassettes targeting Apob, driven by the HI promoter, or targeting the Firefly luciferase gene (Flue), driven by the U6 promoter were inserted adjacent to the mTR (m- R and m-F), within the intron (Intron-R and Intron-F), or adjacent to the wtTR (Wt-R and Wt- F).
• FIG. 2B shows agarose gel analysis of self-complementary AAV vector genomes carrying shApob, driven by the HI promoter, or shFluc (not shown), driven by the U6 promoter.
• FIG. 2C shows vectors depicted in (FIG. 2A) were packaged into AAV9 capsids and assessed for yield by qPCR using an EGFP primer/probe set. Constructs carrying the same shRNA cassette were packaged and titrated as a set (at the same time) to ensure fair comparisons. The two sets of constructs (U6-shFluc and Hl-shApob) were packaged at different times.
• FIG. 3 shows a schematic depiction of 3 generation lentiviral vector production.
• four constructs (packaging plasmid, Rev encoding plasmid, Env encoding plasmid, transgene encoding plasmid) are transfected into a permissive cell line (e.g. , HEK293) to produce the vectors.
• a permissive cell line e.g. , HEK293
• FIG. 4 shows cells infected with a lentiviral vector expressing either GFP (Lenti-GFP, top) or GFP fused to an 80-mer Glycine- Arginine (Lenti-GFP-GRgo) di-amino acid repeat peptide.
• GRgo is a cytotoxic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)-related peptide. Fluorescence imaging shows lower transduction of cells by Lenti-GFP- GRgo compared to Lenti-GFP, indicating lower replication or packaging efficiency of the vector comprising cytotoxic protein (GRgo) relative to vector comprising GFP.
• ALS amyotrophic lateral sclerosis
• FTD frontotemporal dementia
• FIG. 5 shows a schematic depiction of a strategy for increasing replication and/or packaging of a viral vector comprising transgene resistant to packaging (e.g. , a cytotoxic transgene or a transgene that reduces fitness of a host cell).
• Packaging cells are co-transfected with the viral vector production plasmid(s) and a plasmid capable of expressing an interfering RNA molecule (e.g. , shRNA, dsRNA, etc.) specific for the transgene resistant to packaging (e.g. , a cytotoxic transgene or a transgene that reduces fitness of a host cell).
• Transient silencing of transgene expression during packaging e.g. , mediated by RNAi machinery such as Ago2 increases viral vector replication and packaging, leading to an increased yield.
• FIG. 6 shows cells infected with a lentiviral vector expressing either GFP (Lenti-GFP, top), Lenti-GFP-GRgo, or Lenti-GFP-GRgo that was packaged during transient gene expression silencing by a plasmid expressing Lenti-GFP-GRgo-specific shRNA (shRNA-GFP).
• Fluorescence imaging shows lower transduction of cells by Lenti-GFP-GRgo compared to Lenti- GFP, indicating lower replication or packaging efficiency of the vector comprising cytotoxic protein (GRgo) relative to vector comprising GFP.
• GRgo cytotoxic protein
• FIG. 7 shows a schematic depiction of two strategies for increasing replication and/or packaging of a viral vector comprising a transgene resistant to packaging (e.g. , a cytotoxic transgene or a transgene that reduces fitness of a host cell).
• packaging cells are co- transfected with the viral vector production plasmid(s) and a plasmid capable of expressing an shRNA specific for the transgene.
• Transient silencing of transgene expression during packaging e.g. , mediated by RNAi machinery such as Ago2
• increases viral vector replication and packaging leading to an increased yield.
• one or more (e.g. , 3) artificial miRNA (amiRNA) binding sites are engineered into the plasmid comprising transgene resistant to packaging (e.g. , a cytotoxic transgene or a transgene that reduces fitness of a host cell).
• amiRNA artificial miRNA
• Packaging cells are co-transfected with the viral vector production plasmid(s) and a plasmid capable of expressing an amiRNA that is specific for the binding sites engineered into the production plasmid.
• FIG. 8 shows exemplary data demonstrating that RNAi potency increases when an increasing number of miRNA binding sites is incorporated into a transgene construct.
• zero, one, or three miR-122 binding sites were incorporated into a nLacZ expression construct.
• Huh7 cells were transfected with each construct and nLacZ expression was measured.
• Data indicate decreased transgene (nLacZ) expression in cells transfected with constructs having one or three miR- 122 binding sites.
• a similar decrease in transgene expression was also observed in mouse livers.
• FIG. 9 shows specific and efficient gene silencing by the interaction between artificial miRNA (amiRNA) and their target sites.
• Cells were co-transfected with an EGFP construct comprising multiple miRNA binding sites, specific for either 333T or 856T (which are sequences that are not bound by known mammalian miRNAs), and a plasmid expressing either miR-333 or miR-856 amiRNA.
• Data indicate silencing of EGFP-333T expression in cells that were co-transfected with miR-333 amiRNA but not miR-856 amiRNA or a shRNA control plasmid.
• Data indicate silencing of EGFP-856T expression in cells that were co-transfected with miR-856 amiRNA but not miR-333 amiRNA or a shRNA control plasmid.
• FIG. 10 shows representative data relating to methods for improving titer and vector packaging for a lentiviral vector capable of expressing apolipoprotein LI (Apoll), which typically is difficult to package using conventional viral vector production procedures.
• Three miR-856 binding sites (3 x 856T) were incorporated into a Apoll expression construct.
• Packaging cells were co-transfected with the Apoll expression construct and a plasmid expressing amiR-856. Data show that vector titer increases with an increasing amiR-856 concentration, indicating that increased silencing of transgene (e.g., Apoll) expression during packaging increases efficiency of Lenti- Apoll vector production.
• transgene e.g., Apoll
• FIG. 11 shows representative data indicating that apolipoprotein LI expressed by vector (e.g., Lenti- Apoll) that has been packaged during silencing of transgene expression is functional, as indicated by death of cells infected with Lenti- Apoll vector but not control Lenti- GFP vector.
• vector e.g., Lenti- Apoll
• aspects of the disclosure relate, in part, to the discovery that abolishing transgene expression by RNA interference or similar pathway (either shRNAs or artificial miRNAs, amiRNAs) during vector packaging (e.g., packaging of recombinant viral particles in host cell) results in efficient vector production.
• RNA interference or similar pathway either shRNAs or artificial miRNAs, amiRNAs
• lentiviral vectors carrying cytotoxic transgenes e.g., EGFP-(GR)S0 or ApoLl
• cytotoxic transgenes e.g., EGFP-(GR)S0 or ApoLl
• a second strategy for viral vector production is also described herein. Briefly, three copies of target sites for either shRNAs or artificial miRNAs were incorporated into the 3'UTR of ApoLl transgene in the Lenti-ApoLl plasmid. Those target sites were designed not to be recognized by any known mammalian endogenous small RNAs (e.g., the miRNA binding sites are orthogonal with respect to a host cell) but specifically sensitive to the shRNA or amiRNA (e.g., orthogonal shRNA or amiRNA) expressed from a co-transfected plasmid in the packaging process.
• shRNA or amiRNA e.g., orthogonal shRNA or amiRNA
• an "orthogonal" inhibitory nucleic acid or nucleic acid binding site refers to a sequence of an inhibitory nucleic acid (or it' s cognate binding site) that is not naturally expressed in a host cell and does not interact with miRNAs (or miRNA binding sites) endogenously expressed by a host cell. It was observed that production of viral vectors having transgene embedded with the artificial small RNA target sites is not compromised. Instead, transgene expression is efficiently silenced in the presence of the corresponding shRNA or amiRNA (e.g., transiently silenced during viral particle packaging). Accordingly, virus particles comprising a cytotoxic transgene were successfully produced with a high titer. Additionally, it was observed that infection of HEK293 cells with the viral particles caused massive cell death, indicating the infectivity of packaged viral vector and maintenance of transgene function.
• cytotoxic or incompatible transgenes during vector production by transient RNA silencing enables the production of high titer and functional viral vectors.
• Methods described by the disclosure are useful for the packaging of viral vectors such as Adenovirus, lentivirus vectors, adeno-associated virus (AAV), etc. , carrying cytotoxic or incompatible transgenes (e.g. , transgenes that are detrimental to the fitness of a host cell).
• Nucleic acids such as Adenovirus, lentivirus vectors, adeno-associated virus (AAV), etc.
• nucleic acid refers to polymers of linked nucleotides, such as DNA, RNA, etc.
• proteins and nucleic acids of the disclosure are isolated.
• the DNA of a transgene is transcribed into a messenger RNA (mRNA) transcript.
• isolated means artificially produced (e.g. , an artificially produced nucleic acid, or an artificially produced protein, such as a capsid protein).
• isolated means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
• An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.
• PCR polymerase chain reaction
• An isolated nucleic acid may be substantially purified, but need not be.
• a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides.
• Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
• the term "isolated" refers to a protein or peptide that has been artificially produced (e.g.
• a transgene is a nucleic acid sequence, which is not homologous to vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
• a transgene encodes a therapeutic protein or therapeutic functional RNA.
• therapeutic proteins include toxins, enzymes (e.g.
• the nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a cell of a target tissue.
• the disclosure relates to viral vectors encoding one or more transgenes that are cytotoxic or detrimental to the fitness of a host cell.
• a "cytotoxic" transgene refers to a transgene that encodes a gene product (e.g. , a protein) that is toxic to a living cell. Examples of toxic transgenes include transgenes encoding diphtheria toxin, botulinum toxin, ribosome inactivating proteins (e.g. , ricin), cytolysins, porins (e.g. , actinoporins), apolipoproteins, certain proteases, etc. In some embodiments, a protein becomes cytotoxic when overexpressed in a cell.
• transgene that is detrimental to the health of a host cell refers to a transgene encoding a protein having certain physiochemical characteristics (e.g. , a secondary structure having a high thermostability, a tendency to aggregate, etc. ) that results in a reduced fitness (ability to survive) of a host cell expressing that transgene relative to a host cell that does not express the transgene.
• certain physiochemical characteristics e.g. , a secondary structure having a high thermostability, a tendency to aggregate, etc.
• vector includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. , which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
• the term includes cloning and expression vehicles, as well as viral vectors.
• useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter.
• a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
• the phrases “operatively positioned,” “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
• expression vector or construct means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
• expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product (e.g. , a therapeutic protein or therapeutic minigene) or inhibitory RNA (e.g., shRNA, miRNA, amiRNA, miRNA inhibitor) from a transcribed gene.
• a biologically-active polypeptide product e.g. , a therapeutic protein or therapeutic minigene
• inhibitory RNA e.g., shRNA, miRNA, amiRNA, miRNA inhibitor
• Interfering nucleic acid refers to a polymer of linked oligonucleotides which binds and specifically inhibit the expression of a transgene.
• Interfering nucleic acids can be, for example, short-interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), or artificial microRNAs (amiRNA).
• Short hairpin RNAs shRNAs
• shRNAs Short hairpin RNAs
• stem-loop structure that includes a single nucleic acid encoding a stem portion having a duplex comprising a sense strand (e.g. , passenger strand) connected to an antisense strand (e.g. , guide strand) by a loop sequence.
• the passenger strand and the guide strand share complementarity. In some embodiments, the passenger strand and guide strand share 100% complementarity.
• the passenger strand and guide strand share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% complementarity.
• a passenger strand and a guide strand may lack complementarity due to a base-pair mismatch.
• the passenger strand and guide strand of a hairpin-forming RNA have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 at least 8, at least 9, or at least 10 mismatches.
• the first 2-8 nucleotides of the stem are referred to as "seed" residues and play an important role in target recognition and binding.
• the first residue of the stem is referred to as the "anchor” residue.
• hairpin-forming RNA have a mismatch at the anchor residue.
• Hairpin-forming RNA are useful for translational repression and/or gene silencing via the RNAi pathway. Due to having a common secondary structure, hairpin-forming RNA share the characteristic of being processed by the proteins Drosha and Dicer prior to being loaded into the RNA-induced silencing complex (RISC).
• RISC RNA-induced silencing complex
• Duplex length amongst hairpin-forming RNA can vary. In some embodiments, a duplex is between about 19 nucleotides and about 200 nucleotides in length. In some embodiments, a duplex is between about between about 14 nucleotides to about 35 nucleotides in length. In some embodiments, a duplex is between about 19 and 150 nucleotides in length.
• hairpin-forming RNA has a duplex region that is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides in length. In some embodiments, a duplex is between about 19 nucleotides and 33 nucleotides in length. In some embodiments, a duplex is between about 40 nucleotides and 100 nucleotides in length. In some embodiments, a duplex is between about 60 and about 80 nucleotides in length.
• Methods of the current disclosure describe a nucleic acid expressing an interfering nucleic acid, wherein the interfering nucleic acid specifically inhibits expression of a transgene.
• the nucleic acid expresses a shRNA, which will bind and block transcription of the transgene.
• MicroRNAs miRNAs
• Artificial microRNAs amiRNA Artificial microRNAs
• MicroRNAs are small, non-coding RNAs which regulate cellular gene expression by post-transcriptional silencing. When miRNAs are partially complementary to the target mRNA sequences, they typically reduce target mRNA stability and inhibit translation. In contrast, when miRNAs are nearly perfectly complementary to mRNA targets, the mRNA is cleaved, triggering its wholesale destruction. miRNA can achieve tissue specific regulation of systemically delivered and ubiquitously expressed transgenes at post-transcriptional level.
• miRNAs have distinct expression profiles in different tissues and cell types, which differentially regulate transcriptional profiles of cellular genes and cellular functions. Therefore, methods provided herein employ miRNAs to silence transgene expression in cells.
• a miRNA inhibits the function of the mRNAs it targets and, as a result, inhibits expression of the polypeptides encoded by the mRNAs.
• blocking partially or totally
• the activity of the miRNA e.g., silencing the miRNA
• derepression of polypeptides encoded by mRNA targets of a miRNA is accomplished by inhibiting the miRNA activity in cells through any one of a variety of methods.
• blocking the activity of a miRNA can be accomplished by hybridization with a small interfering nucleic acid (e.g., antisense oligonucleotide, miRNA sponge, TuD RNA) that is complementary, or substantially complementary to, the miRNA, thereby blocking interaction of the miRNA with its target mRNA.
• a small interfering nucleic acid that is substantially complementary to a miRNA is one that is capable of hybridizing with a miRNA, and blocking the miRNA' s activity.
• an small interfering nucleic acid that is substantially complementary to a miRNA is an small interfering nucleic acid that is an small interfering nucleic acid that is
• an small interfering nucleic acid sequence that is substantially complementary to a miRNA is an small interfering nucleic acid sequence that is complementary with the miRNA at, at least, one base.
• amiRNAs exploit the miRNA biogenesis pathway described above to produce artificially-designed small RNAs utilizing a miRNA gene backbone.
• the cellular processing of amiRNAs generates a single type of small RNA population which all possess the same selective nucleic acid sequence, which is generally 21 base pairs in length.
• AmiRNAs thereby provide a feasible method for silencing an individual transgene or simultaneously silencing closely-related gene isoforms. AmiRNAs are sometimes advantageous over traditional miRNAs because of higher gene silencing specificity and less off-target silencing effects.
• an artificial microRNA is derived by modifying native miRNA to replace natural targeting regions of pre-mRNA with a targeting region of interest.
• a naturally occurring, expressed miRNA can be used as a scaffold or backbone (e.g. , a pri-miRNA scaffold), with the stem sequence replaced by that of an miRNA targeting a gene of interest (e.g. , an miRNA that is orthogonal to a host cell, for example miR-333 or miR- 856).
• An artificial precursor microRNA pre-amiRNA
• viral vectors and particles disclosed herein e.g. , scAAV vectors and scAAVs described herein
• AmiRNA is derived from a pri-miRNA selected from the group consisting of pri-MIR-21, pri- MIR-22, pri-MIR-26a, pri-MIR-30a, pri-MIR-33, pri-MIR-122, pri-MIR-375, pri-MIR- 199, pri- MIR-99, pri-MIR- 194, pri-MIR- 155, and pri-MIR-451.
• transgenes may be engineered to express a protein of interest, e.g. , a therapeutic protein, and one or more binding sites for an inhibitory nucleic acid (e.g. , shRNA, miRNA, amiRNA, etc.).
• a transgene comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more miRNA binding sites.
• Transcripts expressing such proteins may also be engineered to contain one or more inhibitory miRNAs (e.g. , an miRNA that is not expressed in a host cell). In this way, the transcript, if expressed in a host cell, may be degraded via the inhibitory nucleic acids (e.g.
• a construct engineered to express a transgene in a mammalian cell comprises a transgene having one or more binding sites for an inhibitory nucleic acid (e.g. , miRNA, amiRNA, etc.) that is only endogenously expressed in plant cells or insect cells.
• an inhibitory nucleic acid e.g. , miRNA, amiRNA, etc.
• a construct engineered to express a transgene in an insect cell comprises a transgene having one or more binding sites for an inhibitory nucleic acid (e.g. , miRNA, amiRNA, etc.) that is only endogenously expressed in mammalian cells or plant cells.
• an inhibitory nucleic acid e.g. , miRNA, amiRNA, etc.
• a transcript comprises one or more binding sites for an miRNA selected from miR-333 (SEQ ID NO: 1) or miR-856 (SEQ ID NO: 2).
• a binding site for miR-333 is represented by the sequence set forth in SEQ ID NO: 3.
• a binding site for miR-856 is represented by the sequence set forth in SEQ ID NO: 4.
• inhibitory nucleic acid e.g. , shRNA, miRNA, amiRNA, etc.
• binding sites for an inhibitory nucleic acid are positioned in a 5' untranslated region (5'UTR) of a transcript.
• one or more binding sites for an inhibitory nucleic acid are positioned in an intron.
• one or more binding sites for an inhibitory nucleic acid are positioned between the last codon of a last codon and the polyA tail of the transcript.
• one or more binding sites for an inhibitory nucleic acid are positioned in 5'UTR and one or more binding sites for an inhibitory nucleic acid are positioned between the last codon of a last codon and the polyA tail of a transcript.
• Viral vectors present a powerful tool for the delivery of plasmids and genetic material into cells.
• Adapting plasmid DNA for use with virus -mediated delivery has provided numerous advantages for research, including the delivery of genetic information in traditionally hard-to- transfect cells, such as neurons.
• Viruses naturally infect host cells and direct them to reproduce the viral genome.
• scientists have taken advantage of this process by providing the virus with alternate genomes (e.g., plasmids encoding a nucleic acid or transgene), which can then be replicated once the virus has infected a host cell.
• researchers can introduce plasmids into a host cell to generate recombinant virus.
• viral genomes used in research have been modified through the removal of certain genes that are required for viral replication. These genes are usually divided among numerous "accessory plasmids" which must also be present in the cell for a viral particle to be produced.
• the production of viral particles comprising nucleic acid(s) of interest, along with the viral genome, by a host cell is herein referred to as "packaging".
• the process for the delivery and packaging of nucleic acids into viral genomes varies depending on the viral genome the nucleic acid is encoded in and will be discussed in greater detail for each viral vector below.
• Transgenes expressed from viral genomes for packaging in host cells can be toxic (e.g., cytotoxic or detrimental to the fitness of a host cell, and thus can interfere with viral packaging in the host cell.
• a transgene expressed from the first nucleic acid is cytotoxic to host cells.
• the transgene expressed forms a second structure with high thermal stabilities.
• recombinant virus or “recombinant viral particle” refers to a particle produced in a host cell which encapsulates nucleic acid produced from exogenous DNA inserted into the host cell genome is, has been introduced.
• the disclosure provides transfected host cells.
• transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
• transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor
• nucleotide integration vector and other nucleic acid molecules
• suitable host cells such as a nucleotide integration vector and other nucleic acid molecules.
• a host cell may be transfected with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more isolated nucleic acids.
• Lentiviral vectors are derived from human immunodeficiency virus- 1 (HIV-1).
• HIV-1 human immunodeficiency virus- 1
• the lentiviral genome consists of single- stranded RNA that is reverse-transcribed into DNA and then integrated into the host cell genome. Lentiviruses can infect both dividing and non-dividing cells, making them attractive tools for gene therapy.
• the lentiviral genome is around 9 kb in length and contains three major structural genes: gag, pol, and env.
• the gag gene is translated into three viral core proteins: matrix (MA) proteins, which are necessary for virion assembly and infection of non-dividing cells; capsid (CA) proteins, which form the hydrophobic core of the virion; and nucleocapsid (NC) proteins, which protect the viral genome by coating and associating tightly with the RNA.
• the pol gene encodes for the viral protease, reverse transcriptase, and integrase enzymes which are essential for viral replication.
• the env gene encodes for the viral surface glycoproteins, which are essential for virus entry into the host cell by enabling binding to cellular receptors and fusion with cellular membranes.
• the viral glycoprotein is derived from vesicular stomatitis virus (VSV-G).
• VSV-G vesicular stomatitis virus
• the viral genome also contains regulatory genes, including tat and rev.
• Tat encodes transactivators critical for activating viral transcription, while rev encodes a protein that regulates the splicing and export of viral transcripts.
• Tat and rev are the first proteins synthesized following viral integration and are required to accelerate production of viral mRNAs.
• Methods of the current disclosure describe a recombinant lentiviral transfer vector encoding one or more transgenes of interest flanked by long terminal repeat (LTR) sequences. These LTRs are identical nucleotide sequences that are repeated hundreds or thousands of times and facilitate the integration of the transfer plasmid sequences into the host cell genome.
• LTRs long terminal repeat
• Methods of the current disclosure also describe one or more accessory plasmids.
• These accessory plasmids may include one or more lentiviral packaging plasmids, which encode the pol and rev genes that are necessary for the replication, splicing, and export of viral particles.
• the accessory plasmids may also include a lentiviral envelope plasmid, which encodes the genes necessary for producing the viral glycoproteins which will allow the viral particle to fuse with the host cell.
• the isolated nucleic acids of the disclosure may be recombinant adeno-associated virus
• an isolated nucleic acid as described by the disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof.
• the isolated nucleic acid e.g. , the recombinant AAV vector
• “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs).
• the transgene may comprise, as disclosed elsewhere herein, one or more regions that encode one or more proteins and/or one or more binding sites for inhibitory nucleic acids (e.g. , shRNA, miRNAs, etc.).
• the transgene may also comprise a region encoding, for example, a protein and/or an expression control sequence (e.g. , a poly-A tail), as described elsewhere in the disclosure.
• ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g. , texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)).
• AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types.
• the isolated nucleic acid e.g.
• the rAAV vector comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrhlO, AAVrh39, AAVrh43, AAV2/2-66, AAV2/2-84, AAV2/2-125, and variants thereof.
• the isolated nucleic acid comprises a region (e.g. , a first region) encoding an AAV2 ITR.
• the isolated nucleic acid further comprises one or more AAV ITRs.
• an AAV ITR has a serotype selected from AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrhlO, AAVrh39, AAVrh43, AAV2/2-66, AAV2/2-84, AAV2/2-125, and variants thereof.
• an AAV ITR is a mutant ITR (mTR) that lacks a functional terminal resolution site (TRS).
• the term "lacking a terminal resolution site" can refer to an AAV ITR that comprises a mutation (e.g.
• a sense mutation such as a non- synonymous mutation, or missense mutation
• a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g. , a ATRS ITR).
• TRS terminal resolution site
• a rAAV vector comprising an ITR lacking a functional TRS produces a self-complementary rAAV vector, for example as described by McCarthy (2008) Molecular Therapy 16(10): 1648- 1656.
• scAAV self-complementary AAV vector
• scAAV vectors generate single-stranded, inverted repeat genomes, with a wild-type (wt) AAV TR at each end and a mutated TR (mTR) in the middle.
• isolated nucleic acids comprise DNA sequences encoding RNA hairpin structures (e.g.
• the disclosure provides rAAV (e.g. self-complementary AAV; scAAV) vectors comprising a single-stranded self-complementary nucleic acid with inverted terminal repeats (ITRs) at each of two ends and a central portion comprising a promoter operably linked with a sequence encoding a hairpin-forming RNA (e.g. , shRNA, miRNA, amiRNA, etc.).
• rAAV e.g. self-complementary AAV; scAAV
• ITRs inverted terminal repeats
• the sequence encoding a hairpin-forming RNA is substituted at a position of the self-complementary nucleic acid normally occupied by a mutant ITR.
• Recombinant AAV (rAAV) vectors are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell.
• the transgene is a nucleic acid sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g. , miRNA, miRNA inhibitor) or other gene product, of interest.
• the nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene
• the instant disclosure provides a vector comprising a single, czs-acting wild-type ITR.
• the ITR is a 5' ITR.
• the ITR is a 3' ITR
• ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITR(s) is used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify ITR sequences is within the skill of the art. (See, e.g. , texts such as Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)).
• an ITR may be mutated at its terminal resolution site (TR), which inhibits replication at the vector terminus where the TR has been mutated and results in the formation of a self-complementary AAV.
• TR terminal resolution site
• Another example of such a molecule employed in the present disclosure is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' AAV ITR sequence and a 3 ' hairpin-forming RNA sequence.
• AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types.
• the rAAVs of the disclosure are pseudotyped rAAVs.
• a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with the proteins of Y will be designated as AAVX/Y (e.g. AAV2/1 has the ITRs of AAV2 and the capsid of AAVl).
• pseudotyped rAAVs may be useful for combining the tissue-specific targeting capabilities of a capsid protein from one AAV serotype with the viral DNA from another AAV serotype, thereby allowing targeted delivery of a transgene to a target tissue.
• capsid proteins are structural proteins encoded by the cap gene of an AAV.
• AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing.
• the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa.
• capsid proteins upon translation, form a spherical 60-mer protein shell around the viral genome.
• the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host.
• capsid proteins deliver the viral genome to a host in a tissue specific manner.
• an AAV capsid protein is of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrhlO, AAVrh39, AAVrh43, AAV2/2-66, AAV2/2-84, AAV2/2- 125.
• an AAV capsid protein is of a serotype derived from a non-human primate, for example scAAV.rh8, AAV.rh39, or AAV.rh43 serotype.
• an AAV capsid protein is of an AAV9 serotype.
• the components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans.
• any one or more of the required components e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions
• a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.
• a stable host cell will contain the required component(s) under the control of an inducible promoter.
• the required component(s) may be under the control of a constitutive promoter.
• a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters.
• a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.
• the recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector).
• the selected genetic element may be delivered by any suitable method, including those described herein.
• the methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g. , Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.
• the adenovirus genome is a non-enveloped, large (36-kb) double-stranded DNA
• dsDNA molecule comprising multiple, heavily spliced transcripts.
• Adenoviruses have high packaging capacity ( ⁇ 8 kilobases) and are able to target a broad range of dividing and non- dividing cells. Adenoviruses do not integrate into the host genome and thus only produce transient transgene expression in host cells. At either end of adenoviral genome are inverted terminal repeats (ITRs). Genes encoded by the adenoviral genome are divided into early (El- E4) and late (L1-L5) transcripts. Most human adenoviral vectors are based on the Ad5 virus type, which uses the Coxsackie-Adenovirus Receptor to enter cells.
• Recombinant adenovirus has the El and E3 genes deleted from its genome. Deletion of
• El renders the virus replication incompetent; El is supplied by adenovirus packaging cell lines, such as HEK293 cells. E3 is involved in evading host cell immunity and is thus not essential for virus production. Deletion of these two components results in a transgene packaging capacity of > 8 kilobases.
• Methods of the current disclosure describe recombinant adenoviral vectors encoding nucleic acid(s) of interest.
• Generation of recombinant adenoviral vectors involves both a transfer vector and an adenoviral vector.
• the transgene to be packaged in adenovirus is initially placed in a transfer vector.
• Recombinant transfer vectors containing left and right flanking sequences which are complementary to the sequences at the site of insertion in the adenoviral genome facilitate insertion of the transgene into the adenoviral plasmid by homologous recombination (HR).
• the left and right sequences are used as templates to repair a DNA DSB in HR, thereby facilitating error-free insertion of the transgene into the adenoviral plasmid.
• Methods of the current disclosure describe the use of one or more accessory plasmids in a host cell.
• the accessory plasmid is a packaging plasmids which encodes all necessary components for viral packaging except the El and the E3 genes.
• An additional accessory plasmid is required to provide the El gene to the packaging cells.
• Retrovirus (most commonly, ⁇ -retrovirus) is an RNA virus comprised of the viral genome and several structural and enzymatic proteins, including reverse transcriptase and integrase. Once inside a host cell, the retrovirus uses the reverse transcriptase to generate a DNA provirus from the viral genome. The integrase protein then integrates this provirus into the host cell genome for production of viral genomes encoding the nucleic acid(s) of interest. Retrovirus can package relatively high amounts of DNA (up to 8 kilobases), but are unable to infect non-dividing cells and insert randomly into the host cell genome.
• Retroviral transfer and packaging vectors are similar to the lentiviral system described above.
• Retroviral transfer vectors typically encode a nucleic acid of interest flanked by LTRs, which are derived from Moloney Murine Leukemia Virus (MoMLV) or Murine Stem Cell Virus (MSCV) sequences.
• LTRs which are derived from Moloney Murine Leukemia Virus (MoMLV) or Murine Stem Cell Virus (MSCV) sequences.
• Methods of the current disclosure describe the use of one or more accessory plasmids.
• the accessory plasmids are a packaging plasmid, which encodes the gag and pol genes, and an envelope plasmid which encodes the env gene.
• the gag gene is translated into three viral core proteins: matrix (MA) proteins, which are necessary for virion assembly and infection of non-dividing cells; capsid (CA) proteins, which form the hydrophobic core of the virion; and nucleocapsid (NC) proteins, which protect the viral genome by coating and associating tightly with the RNA.
• matrix (MA) proteins which are necessary for virion assembly and infection of non-dividing cells
• capsid (CA) proteins which form the hydrophobic core of the virion
• NC nucleocapsid
• the pol gene encodes for the viral protease, reverse transcriptase, and integrase enzymes which are essential for viral replication.
• a "host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest or of packaging the nucleic acid of interest into a viral particle. Often a host cell is a mammalian cell. Examples of host cells include human cells, mouse cells, rat cells, dog cells, cat cells, hamster cells, monkey cells, insect cells, plant cells, or bacterial cells.
• insect cells include but are not limited to Spodoptera frugiperda ⁇ e.g., Sf9, Sf21), Spodoptera exigua, Heliothis virescens, Helicoverpa zea, Heliothis subflexa, Anticarsia gemmatalis, Trichopulsia ni ⁇ e.g., High-Five cells), Drosophila melanogaster ⁇ e.g., S2, S3), Antheraea eucalypti, Bombyx mori, Aedes alpopictus, Aedes aegyptii, and others.
• Examples of bacterial cells include, but are not limited to Escherichia coli, Corynebacterium glutamicum, and Pseudomonas fluorescens.
• yeast cells include but are not limited to Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris, Bacillus sp., Aspergillus sp., Trichoderma sp., and Myceliophthora thermophila CI.
• Examples of plant cells include but are not limited to Nicotiana sp.,
• Arabidopsis thaliana Mays zea, Solarium sp., or Lemna sp.
• a host cell is a mammalian cell.
• mammalian cells include Henrietta Lacks tumor (HeLa) cells and baby hamster kidney (BHK-21) cells.
• a host cell is a human cell, for example a HEK293T cell.
• a host cell may be used as a recipient of one or more viral transfer vectors and one or more accessory plasmids.
• the term includes the progeny of the original cell which has been transfected.
• a "host cell” as used herein may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
• cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
• the terms "recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.
• compositions and methods for improving vector yield, viral titer, and/or recombinant viral particle (e.g. , rAAV particle) production in host cells relate to compositions and methods for improving vector yield, viral titer, and/or recombinant viral particle (e.g. , rAAV particle) production in host cells.
• methods described by the disclosure improve vector yield, viral titer, and/or recombinant viral particle (e.g. , rAAV particle) by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, or more (e.g. , 20-fold, 100-fold, 200-fold, 1000-fold, or more) relative to methods of viral particle production that do not employ pairs of orthogonal (with respect to a host cell) inhibitory nucleic acids and cognate binding sites.
• FIG. 1 provides several non- limiting examples of gene expression vectors.
• transgenes e.g. , transgene products that are toxic to packaging cells or incompatible with viral vector packaging systems
• Possible causes of low titer, or no production of viral vectors include, for example, bad quality of vector constructs, mutations or deletions of packaging signal/replication origin in vector constructs, secondary structure with high thermal stabilities that hinder vector genome replication, or other factors (e.g. , cytotoxic transgenes, or transgenes that interfere with vector genome replication or packaging).
• FIG. 2A shows a schematic of self- complementary AAV (scAAV) plasmids comprising shRNA cassettes targeting Apob, driven by the HI promoter, or targeting the Firefly luciferase gene (Flue), driven by the U6 promoter.
• scAAV self- complementary AAV
• the cells were transformed with the expression constructs and agarose gel analysis was performed. Truncated viral genomes were observed in lanes for all shRNA cassettes but not in control cassette (no shRNA) (FIG. 2B). Vectors were packaged into AAV9 capsids and assessed for yield by qPCR using an EGFP primer/probe set. Data indicate a lower AAV yield when shRNA encoding sequences are in close proximity to the wild-type ITR (wtTR) of the construct (FIG. 2C).
• the secondary structure of a transgene compromises packaging or yield of viral vectors, such as lentiviral vectors.
• packaging cells e.g. , HEK293T
• four plasmids a packaging plasmid comprising the gag gene, an Env-encoding plasmid, a Rev-encoding plasmid, and a vector plasmid comprising a transgene.
• Transgene cytotoxicity may result in reduced viral replication and/or packaging efficiency, for example in the context of lentiviral vectors. Therefore, production of two lentiviral vectors carrying cytotoxic transgenes (e.g. , EGFP-(GR) 8 o (ALS/ FTD-related dipeptide repeat protein) or Apoll (apolipoprotein LI)) was investigated. Cells were infected with a lentiviral vector expressing either GFP (Lenti-GFP, top) or GFP fused to an 80-mer Glycine- Arginine (Lenti-GFP-GR 8 o) di-amino acid repeat peptide.
• cytotoxic transgenes e.g. , EGFP-(GR) 8 o (ALS/ FTD-related dipeptide repeat protein) or Apoll (apolipoprotein LI)
• Fluorescence imaging shows lower transduction of cells by Lenti-GFP-GR 8 o compared to Lenti-GFP, indicating lower replication or packaging efficiency of the vector comprising cytotoxic protein (GR 8 o) relative to vector comprising GFP (FIG. 4).
• FIG. 5 A strategy for increasing replication and/or packaging of a viral vector comprising an un- packagable (e.g. , cytotoxic) transgene was developed (FIG. 5). Briefly, packaging cells are co- transfected with the viral vector production plasmid(s) and a plasmid capable of expressing an interfering RNA molecule (e.g. , shRNA, dsRNA, etc.) specific for the unpackageable (e.g. , cytotoxic) transgene. Transient silencing of transgene expression during packaging (e.g. , mediated by RNAi machinery such as Ago2) increases viral vector replication and packaging, leading to an increased yield.
• an interfering RNA molecule e.g. , shRNA, dsRNA, etc.
• Cells were infected with a lentiviral vector expressing either GFP (Lenti-GFP, FIG. 6 top), Lenti-GFP-GRgo (FIG. 6, middle), or Lenti-GFP-GRgo that was packaged during transient gene expression silencing by a plasmid expressing Lenti-GFP-GRgo-specific shRNA (FIG. 6, bottom). Fluorescence imaging shows lower transduction of cells by Lenti-GFP-GRgo compared to Lenti-GFP, indicating lower replication or packaging efficiency of the vector comprising cytotoxic protein (GRgo) relative to vector comprising GFP.
• GRgo cytotoxic protein
• RNAi potency increases when an increasing number of miRNA binding sites is incorporated into a transgene construct. For example, zero, one, or three miR- 122 binding sites were incorporated into a nLacZ expression construct. Huh7 cells were transfected with each construct and nLacZ expression was measured. Data indicate decreased transgene (nLacZ) expression in cells transfected with constructs having one or three miR- 122 binding sites (FIG. 8). A similar decrease in transgene expression was also observed in mouse livers (FIG. 8).
• FIG. 9 shows specific and efficient gene silencing by the interaction between artificial miRNA (amiRNA) and their target sites.
• Cells were co-transfected with an EGFP construct comprising multiple miRNA binding sites, specific for either 333T or 856T (which are sequences that are not bound by known mammalian miRNAs), and a plasmid expressing either miR-333 or miR-856 amiRNA. Sequences of miR-333 and miR-856, and their respective binding sites are shown below in Table 1.
• miR-856 binding site GCTGAAGTTATTGGTAGGATTATGCTGAAGTTATTGGTAGGATT 4
• Data indicate silencing of EGFP-333T expression in cells that were co-transfected with miR-333 amiRNA but not miR-856 amiRNA or a shRNA control plasmid.
• Data indicate silencing of EGFP-856T expression in cells that were co-transfected with miR-856 amiRNA but not miR-333 amiRNA or a shRNA control plasmid.
• a second strategy for increasing replication and/or packaging of a viral vector comprising an un-packagable (e.g. , cytotoxic) transgene was also developed (FIG. 7). Briefly, one or more (e.g. , 3) artificial miRNA (amiRNA) binding sites are engineered into the plasmid comprising the un-packagable (e.g. , cytotoxic) transgene.
• an un-packagable e.g. , cytotoxic
• Packaging cells are co-transfected with the viral vector production plasmid(s) and a plasmid capable of expressing an amiRNA that is specific for the binding sites engineered into the production plasmid.
• the amiRNA bind to the target sites located on the transgene and transiently silence transgene expression, resulting in improved viral titer and packaging efficiency.
• a lentiviral vector capable of expressing apolipoprotein LI (Apoll), which typically cannot be packaged using conventional viral vector production procedures, was produced using the strategy outlined in FIG. 7. Briefly, three miR-856 binding sites (3 x 856T) were incorporated into a Apoll lentivirus expression construct. Packaging cells were co-transfected with the Apoll expression construct and a plasmid expressing amiR-856 (FIG. 10, left). Data show that vector titer increases with an increasing amiR-856 concentration, indicating that increased silencing of transgene (e.g. , Apoll) expression during packaging increases efficiency of Lenti- Apoll vector production (FIG. 10, right). Additionally, data indicate that
• apolipoprotein LI expressed by the vector e.g. , Lenti- Apoll
• the vector e.g. , Lenti- Apoll
• apolipoprotein LI expressed by the vector is functional, as evidenced by death of cells infected with Lenti-Apoll vector but not control Lenti-GFP vector (FIG. 11).
• the data described in this example demonstrate that co-transfection of transient suppression of transgene expression during the packaging phase of viral vector production successfully generated high titer and functional viral (e.g. , lentiviral) vectors, even when such vectors comprise cytotoxic or otherwise unpackageable transgenes.
• high titer and functional viral e.g. , lentiviral

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