WO2022236174A1

WO2022236174A1 - In situ car-t therapies, vectors and methods therefor

Info

Publication number: WO2022236174A1
Application number: PCT/US2022/028353
Authority: WO
Inventors: David T. Curiel
Original assignee: Washington University
Priority date: 2021-05-07
Filing date: 2022-05-09
Publication date: 2022-11-10

Abstract

The present disclosure provides a system for the in vivo modification of T cells. The system comprises pools of adenovirus particles that have been modified so that they target T- cells. One pool of adenovirus particles comprises an expression cassette encoding a CAR that recognizes a cancer-associated antigen, flanked by polynucleotide arms for CRISPR-mediated insertion of the expression cassette into a T-cell genome. Other pools of adenovirus particles express components required for CRISPR-mediated insertion of the expression cassette. Administration of the system to an individual results in in vivo modification of T-cells so that they express the CAR. Also provided herein are individual adenoviral vectors and targeted adenoviral particles comprising such vectors, for practicing methods of the disclosure.

Description

IN SITU CAR-T THERAPIES, VECTORS AND METHODS THEREFOR

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63/185,916 filed May 7, 2021, which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under UG3TR002851 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Chimeric antigen receptor (CAR) T-cell (CAR T-cell) therapy, such as CAR-T cancer immunotherapy, is a rapidly evolving approach to treatment of diseases such as cancer. Currently, CAR-T methods for cancer immunotherapy involve a number of steps, including obtaining anti -tumor T-cells from a subject such as a cancer patient, modifying the cells in vitro to express one or more antigens, culturing the cells ex vivo , and returning the modified cells to the subject. In these methods, T-cells are acquired from the body of a subject. The cells are genetically modified ex vivo with either a Chimeric Antigen Receptor (CAR) and/or a specific T-cell receptor (TCR), and the modified cells are grown in vitro. When the cells are grown in sufficient numbers, they are administered to the subject. These methods can be difficult, expensive, cumbersome and/or hazardous. The requirements for obtaining, modifying, culturing and administering a subject’s T-cells present major barriers to developing CAR T therapies for cancers and other diseases.

[0004] Similar approaches can also be applied to other immune cells. Such cells include B lymphocytes and NK cells. In each of these instances, the same advantages would accrue the in-situ approach compared to extracorporeal modification as detailed for T-cells. In each of these cases as well, engineered modifications of adenoviral vectors are critical to actualize such an in-situ approach. It is also noteworthy that in vivo modification of T-cell, B cells and NK cells provides the foundation for a range of other gene therapies, over-and-above the CAR-T approach. Herein we provide data showing our ability to achieve in vivo gene transfer to T-cells, B cells, and NK cells. [0005] Compositions and methods are needed for CAR-T therapies that do not require removal of T-cells from the subject’s body or in vitro culturing of a subject’s T- cells.

SUMMARY

[0006] The present disclosure provides a method that utilizes gene-editing technology, such as CRISPR, to modify T-cells within a cancer patient, so that the T-cell recognize the patient’s cancer. Such modification may be achieved using pools of different T-cell targeted adenovirus particles, one pool comprising a nucleic acid sequence encoding a CAR that recognize a cancer antigen, and at least one other pool expressing CRISPR components. Thus, one aspect is a system for in vivo, CRISPR-mediated production of CAR T cells, the system comprising: a) a first targeted adenovirus particle that comprises a first adenovirus vector comprising an expression cassette flanked by a 5’ polynucleotide arm and a 3’ polynucleotide arm; wherein the 5’ polynucleotide arm is homologous to a first polynucleotide sequence in a safe harbor locus of a T cell; wherein the 3’ polynucleotide arm is homologous to a second polynucleotide sequence in the safe harbor locus; and, wherein the expression cassette comprises a heterologous nucleic acid molecule comprising a tissue- specific promoter operably linked to a first heterologous nucleic acid sequence encoding a CAR protein; and, b) a second targeted adenovirus particle that comprises a second adenovirus vector comprising: i) a second heterologous nucleic acid molecule comprising a first mammalian promoter operably linked to a nucleic acid sequence encoding a Cas protein; and, optionally, ii) a third heterologous nucleic acid molecule comprising a second mammalian promoter operably linked to a nucleic acid sequence encoding gRNA that recognizes a third polynucleotide sequence in the safe harbor locus. In some aspects, the second targeted adenovirus particle may not comprise the third heterologous nucleic acid molecule, and the system may comprise a third targeted adenovirus particle comprising a third adenovirus vector, the third adenovirus vector comprising a heterologous nucleic acid molecule comprising a second mammalian promoter operably linked to a nucleic acid sequence encoding gRNA that recognizes a third polynucleotide sequence in the safe harbor locus.

[0007] One aspect is use of a system of the disclosure to treat cancer in an individual. Such use may comprise administering to the individual a system of the disclosure, which may comprise one of more targeted adenovirus particles of the disclosure. [0008] On aspect is a targeted adenovirus particle comprising an adenovirus vector comprising an expression cassette flanked by a 5’ polynucleotide arm and a 3’ polynucleotide arm; wherein the 5’ polynucleotide arm is homologous to a first polynucleotide sequence in a safe harbor locus of a T cell; wherein the 3’ polynucleotide arm is homologous to a second polynucleotide sequence in the safe harbor locus; and, wherein the expression cassette comprises a heterologous nucleic acid molecule comprising a tissue- specific promoter operably linked to a heterologous nucleic acid sequence encoding a CAR protein.

[0009] In these aspects, the targeted adenovirus particles may be targeted to T cells. In these aspects, each of the targeted adenovirus particles may comprise a modified fiber and/or a modified hexon. In these aspects, the modified fiber may increase the T cell infectivity of the targeted adenovirus particle comprising the modified fiber. In these aspects, the modified fiber may comprise a modification to the H1 loop. In these aspects, the modified fiber may comprise insertion of a heterologous targeting ligand, which may be from a serotype of adenovirus that differs from the serotype of adenovirus from which the unmodified fiber originated. In these aspects, the modified fiber may comprise a knob domain from a serotype of adenovirus that differs from the serotype of adenovirus from which the unmodified fiber originated. In these aspects, the knob domain of the modified fiber may be replaced with a fiber knob domain from a serotype of adenovirus that differs from the serotype of adenovirus from which the unmodified fiber originated. In these aspects, the modified fiber may comprise an Ad5 fiber in which the knob domain has been replaced with the knob domain from the fiber of an NADC-1 strain of porcine adenovirus type 4. In these aspects, the knob domain of the modified fiber may comprise and RGD domain. In these aspects, the modified hexon may have reduced affinity for coagulation factor X relative to the affinity of an unmodified hexon for coagulation factor X. In these aspects, the modified hexon may comprise a modification of hypervariable region 7 (HVR7). In these aspects, the modified hexon may be a chimeric hexon, in which HVR7 of the modified hexon may be from a serotype of adenovirus that differs from the serotype type of adenovirus from which the unmodified hexon originated. In these aspects, the modified hexon may comprise an Ad5 hexon in which HVR7 has been replaced with HVR7 from adenovirus serotype 3 ( Ad3).

[0010] One aspect is an adenovirus vector comprising an expression cassette flanked by a 5’ polynucleotide arm and a 3’ polynucleotide arm; wherein the 5’ polynucleotide arm is homologous to a first polynucleotide sequence in a safe harbor locus of a T cell; wherein the 3’ polynucleotide arm is homologous to a second polynucleotide sequence in the safe harbor locus; and, wherein the expression cassette comprises a heterologous nucleic acid molecule comprising a tissue-specific promoter operably linked to a heterologous nucleic acid sequence encoding a CAR protein.

[0011] In any aspect of the disclosure, the safe harbor locus may be selected from the group consisting of the Rogi 1 locus, the Rogi2 locus, and the Rosa26 locus. In any aspect, the tissue specific promoter may be selected from the group consisting of dLck promoter and a CD36 promoter. In any aspect, the CAR may recognize a cancer-associated antigen, which may be EphA2. In any aspect, the cancer may be ovarian cancer. In any aspect, the adenoviral particles may be serotype 5 adenoviral particles, while the adenoviral vectors may be serotype 5 adenoviral vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGS. 1 A & 1B. illustrate a general adenoviral vector and a modified adenovirus particle. FIG. 1 A illustrates a general adenovirus vector comprising an expression cassette comprising a tissue-specific promoter (TSP) and a CAR gene. FIG. IB illustrates a modified adenovirus Type 5 particle highlighting the fiber and knob domain. One knob domain is enlarged to illustrate the HI loop that may be modified to alter tropism of the virus. FIG. IB also highlights hexon proteins in which HVR7 is replaced with HRV7 from Ad3.

[0013] FIGS. 2A-2E shows examples of vectors of the disclosure. FIG. 2A is a schematic illustration of an adenovirus vector comprising an adenovirus genome. FIGS. 2B-2D illustrate editing vectors of the disclosure. FIG. 2B illustrates an adenovirus vector comprising a Cas9 gene under the control of a CMV promoter and a gene encoding a gRNA under the control of a U6 promoter. FIG. 2C illustrates the vector of FIG. 2B but lacking the U6-gRNA expression cassette, while FIG. 2D illustrates the vector of FIG. 2B but lacking the Cas9 expression cassette. FIG. 2E illustrates a donor vector comprising a pCD35-CAR expression cassette flanked by Rosa26 arms.

[0014] FIG. 3 illustrates selective expression in vascular endothelium using an endothelium targeted adenovirus. Epithelial cells were exposed to either wt adenovirus (Ad5; top left) or adenovirus in which the fiber knob domain contained a myeloid binding protein (Ad5.MBP; op right). Epithelial cells were also exposed to adenovirus containing a luciferase gene linked to a CMV promoter (Ad5.CMV) or a R0B04 promoter (Ad5.R0B04).

[0015] FIG. 4 demonstrates that type 5 adenovirus (Ad5) can infect the leukocytes of mice expressing human coxsackie and adenovirus receptor (hCAR mice). FIG. 4A outlines the study protocol. Briefly, leukocytes were purified from the spleens of wt and hCAr +/- mice, which express adenovirus receptor. The purified leukocytes were transduced with a recombinant Ad5 expressing green fluorescent protein (eGFP) and leukocytes examined for expression of GFP. FIG. 4B shows a graphic illustrating the recombinant viruses used in the study. FIG. 4C shows the level of fluorescence obtained at various multiplicities of infection (MOIs).

[0016] FIGS. 5A-5D demonstrates that targeted Ad5 can infect T-cells. FIG. 5A outlines the study protocol. FIG. 5B shows a graphic illustrating the recombinant viruses used in the study. FIG. 5C shows sorting by GFP expression of T-cells transduced with Ad5 expressing GFP and either Ad5 fiber or Ad5 fiber containing the knob region from an NADC-l strain of porcine adenoviais type 4 (Ad5PK4) adenovirus. FIG. 5D shows the percent of T-cells expressing GFP following transduction with either Ad5 or Ad5PK4.

[0017] FIGS. 6A-6C demonstrates that targeted Ad5 can infect T-cells. FIG. 5A outlines the study protocol. FIG. 5B shows a graphic illustrating the recombinant viruses used in the study. FIG. 5C shows the percent of T cells from the spleens of mice treated with either PBS, or Ad5 expressing GFP and either wt Ad5 fiber (Ad5) or fiber containing the knob region from porcine adenovirus type 4 (AdPK4), that express eGFP.

[0018] FIGS. 7 A & 7B illustrate in vivo gene editing of T-lymphocytes via tropism modified adenovirus. FIG. 7A outlines the protocol for the study. FIG. 7B shows results from PCR analyses of T-cells from mice treated with a tropism modified adenovirus carrying donor DNA (AdRGD donor) and a tropism modified adenovirus carrying CRISPR components (AdRGD CRISPR).

DETAILED DESCRIPTION

[0019] CAR T-cell therapy has shown tremendous potential for treating cancers. However, current protocols for producing CAR T-cells are cumbersome and time consuming. The present disclosure provides methods of CAR T-cell production that are less invasive to the patient, less time consuming and more efficient. Specifically, the present disclosure provides a method of producing CAR T-cells in vivo. The method comprises using gene-editing technology, such as CRISPR, to modify T-cells in a cancer patient, so that the T-cell recognize the patient’s cancer. Modification of the in vivo T-cells is accomplished using different pools of targeted adenovirus particles, one pool comprising a nucleic acid sequence encoding a CAR that recognize a cancer antigen, and at least one other pool expressing CRISPR components. Thus, a method of the disclosure may generally be performed by providing to an individual having cancer: i) a targeted adenovirus particle comprising an adenoviral vector, the adenoviral vector comprising an expression cassette containing a CAR-encoding nucleic acid sequence operably linked to a tissue-specific promoter; and, ii) one or more additional adenovirus particles, each additional particle comprising an adenovirus vector that comprises a modified adenoviral genome encoding one or more CRISPR components (e.g., CRISPR associate protein (CAS) (e.g., CAS1, CAS, 2, CAS9), guide RNA (gRNA)). In such methods, the adenovirus particles maybe modified so that they have reduced hepatotropism and increased infectivity for T-cells. Consequently, provision of such components to an individual results in in vivo modification of T-cells such that the modified T-cells express a CAR that can recognize cancer cells.

[0020] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the claims.

[0021] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly, the terms “comprising”, “including” and “having” can be used interchangeably. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0022] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Terms and phrases, which are common to the various aspects disclosed herein, are defined below.

[0024] The term “adenovirus” and “adenoviral” may be used interchangeably herein and have their standard meaning in the art meaning all viruses of the family Adenoviridae, including any adenovirus that infects a human or an animal, further including all groups, subgroups, and serotypes. Depending upon the context, reference to “adenovirus” can refer, for example, to viral particles, vectors, nucleic acid molecules and sequences, and the like. There are at least 51 serotypes of adenovirus that are classified into several subgroups. For example, subgroup A includes adenovirus serotypes 12, 18, and 31. Subgroup C includes adenovirus serotypes 1, 2, 5, and 6. Subgroup D includes adenovirus serotypes 8, 9, 10, 13, 15, 17, 19, 19p, 20, 22-30, 32, 33, 36-39, and 42- 49. Subgroup E includes adenovirus serotype 4. Subgroup F includes adenovirus serotypes 40 and 41.

[0025] As used herein, “^'adenovirus particle”, “adenoviral particle”, and the like, are used interchangeably to refer to infectious viral particles that are formed by encapsulation of an adenoviral vector of the disclosure, with adenovirus capsid proteins comprising, at least, adenoviral hexon protein (“hexon”), adenoviral penton protein (“penton”), and adenoviral fiber protein (“fiber”). Thus, the resulting particle represents a “packaged” adenoviral vector. The resulting viral particles have a variety of uses, including, but not limited to, transferring nucleic acids into cells either in vitro (e.g., cell culture) or in vivo (e.g., in an individual). A vims, or particle, may refer to a single particle, or a stock of particles. The term “adenoviral particie(s)” includes ail viruses that can be categorized as an adenovirus, including any adenovirus that infects a human or an animal, including all groups, subgroups, and serotypes. In some aspects, an adenovirus particle may be an adenovirus 5 (Ad5) adenovirus particle, which means that the hexon, penton, and fiber proteins are from Ad5. "‘Adenovirus particles” includes particles comprising wild type (wt) capsid proteins or capsid proteins that have been modified in various ways disclosed herein.

[0026] “Targeted adenovirus particle” means an adenovirus particle that has been modified so that it preferentially binds a specific type of cell (aka the target cell), such as a T-cell, over another type of cell. “T-cell targeted adenovirus particle” refers to an adenovirus particle that has been modified in such a way that its binding affinity for T-cells is increased relative to an adenovirus particle that has not been so modified (e.g,, a wild type (wt) adenovirus particle). Binding of an adenoviais particle to a cell Is generally mediated by an elongated fiber projecting from each of the fivefold capsid vertices. The proximal end of the fiber is attached to the penton base of the capsid. The distal end of the fiber forms a globular “knob" domain, which generally functions as the attachment site for cellular receptors. Modification of receptor binding sites (aka the targeting ligand) in the knob domain can alter the affinity of the targeting ligand for cell-expressed receptors, and thus, alter the affinity of the adenovirus particle for different cell types. Such modifications may be made in the HI loop or the C loop of the knob domain and may include, for example, insertions, deletions, and/or substitutions of one or more amino acid residues within the loop. In some aspects, the targeting ligand from the fiber of one serotype of adenovirus may be swapped into the corresponding location in the fiber of another serotype of adenovirus, which may alter the tropism of the adenovirus particle. In some aspects, the knob domain in the fiber of one serotype of adenovirus may be replaced with the knob domain from the fiber of another serotype of adenovirus. In one aspect, a targeted adenovirus particle may be an Ad5 particle and comprise an Ad5 fiber in which the knob domain of the Ad5 fiber (e.g., amino acid residues 400 to 582) bas been replaced with the knob domain from the fiber of a serotype 4 adenovirus (e.g., amino acid residues 120 to 703). in some aspects, the serotype 4 adenovirus may be an NADC-i strain of porcine adenovirus type 4. In some aspects, the fiber may be modified so that the knob domain comprises an RGD binding motif, which binds integrins. Thus, in some aspects, the modified fiber comprises an RGD binding domain. In some aspects, a universal acceptor domain (UAD) (e.g., S. aureus protein A, biotin acceptor protein (bap), FLAG peptide) may be inserted into the fiber. The UAD may be recognized by a bridging molecule that also recognizes a cellular receptor and facilitates interaction between the adenovirus and a cellular target. In some aspects, the affinity of a targeted adenovirus particle for the target cell is at least 2X, 5X, 10X, I00X, or 1000X greater than its affinity for a non-target cell. In some aspects, the affinity of a targeted adenoviais particle for the target cell is at least 1 log (10¹), 2 logs, 3 logs, 4 logs, 5 logs, or at least 6 logs greater than its affinity for a non -target cell.

[0027] Adenovirus particles of the disclosure may contain other modifications that improve targeting of the adenovirus particles to T-ceils. For example, it is well known that coagulation factor X (“factor X”) interacts with adenovirus serotype 5 hexon protein, acting as a bridge to bind heparin sulfate proteoglycans, which leads to substantia] hepatocyte uptake. Thus, modifying Ad5 hexon to disrupt its interaction with factor X may help reduce hepatotropism. Such modifications may comprise a hypervariable region (HVR). In some aspects, the modified hexon may comprise an alteration to HVR7. In some aspects, the modified hexon may be produced by replacing HVR7 with a HVR from another serotype of adenovirus. In some aspects, the modified hexon may be produced by replacing HVR7 of Ad5 hexon with HVR.7 from adenovirus serotype 3 (Ad3). In some aspects, the modified hexon may comprise an Ad5 hexon in which HVR7 is replaced with HVR7 from adenovirus serotype 3 (Ad3). In some aspects, the modified hexon may comprise an Ad5 hexon in which the amino acid region from about amino acid 382 to about amino acid 588 is replaced with the corresponding region of hexon from Ad3.

[0028] As used herein, the terms "vector, " "polynucleotide vector, " "polynucleotide vector construct," "nucleic acid vector construct," and "vector construct" are used interchangeably herein to mean any nucleic acid construct that can be used for gene transfer, gene expression, and the like, as understood by those skilled in the an.

[0029] As used herein, the term "viral vector" is used according to its art- recognized meaning and refers to a nucleic acid vector construct that includes at least one element of viral origin and can be packaged into a viral particle, “Adenovirus vector", "adenoviral vector", and the like, may be used Interchangeably and are well understood In the art to mean a nucleic acid vector containing all or a portion of an adenovirus genome. An adenoviral vector refers to nucleic encoding a complete adenoviais genome or a modified adenovirus genome, including one that can be used to introduce heterologous nucleic acid when transferred into a cell, particularly when packaged as a particle. A modified adenovirus genome refers to an adenovirus genome in which alterations have been made to the sequence of the genome. Such alteration may include point mutations, deletions, and insertions. Deletions may include deletion of an entire adenovirus gene, such as El and/or E3 or portions thereof. Insertions may include insertion of a heterologous nucleic acid molecule into the adenovirus genome such that the heterologous nucleic acid molecule is inserted within an adenovirus gene (e.g., E1 or E3), thereby replacing the entire gene or at least a portion thereof. An adenoviral vector may be in any of several forms, including, but not limited to, for example, naked vector DNA, vector ON A associated with a carrier molecule, or vector DNA encapsulated in an adenovirus capsid. A variety of vectors with different requirements and purposes are described herein. For example, one vector may be used to deliver particular nucleic acid molecules into a packaging cell line for stable integration into a chromosome. Other vectors may encode CR1SPR elements that assist with integration of particular nucleic acid molecules.

[0030] A '"chimeric antigen receptor" (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen, such as an antigen expressed by eancer/tumor cells. Generally, a CAR is designed for a T-cell and is a chimera of an intracellular signaling domain of a T-cell receptor (TCR) complex and an extracellular antigen-recognizing domain (e.g., an antibody single chain variable fragment (scFv) or other antigen binding fragment). In some aspects, CAR may also comprise an extracellular hinge domain A T-cell that expresses a CAR may be referred to as a CAR T-cell .

[0031] At least four generations of CARs have been developed, each of which contains different components. First generation CARs join an antibody-derived scFv to the CD3zeta (z or z) intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains. Second generation CARs incorporate an additional domain, e.g., CD28, 4- IBB (4 IBB), or ICOS, to supply a costimulatory signal. Third-generation CARs contain two costimulatory domains fused with the TCR CD3z chain. Third-generation costimulatory domains may include, e.g., a combination of CD3z, CD27, CD28, 4- IBB, ICOS, or 0X40. CARs, in some embodiments, contain an ectodoraain (e.g., CD3z), commonly derived from a single chain variable fragment (scFv), a hinge, a transmembrane domain, and an endodomain with one (first generation), two (second generation), or three (third generation) signaling domains derived from CD3z and/or eo- stimulatory’ molecules. CARs used in compositions and methods of the disclosure may be from any generation of CAR, such as first-generation CARs, second generation CARs, third generation CARs, or combinations or variants thereof. CARs used in compositions and methods of the disclosure may also be humanized or fully human, or be a chimeric molecule comprising a combination of human sequences and sequences from another species (e.g., mouse). In some aspects, the CAR may comprise the EphA2 scFv. In some aspects, the CAR. may comprise the 4-1BB internal domain. [0032] “Operably linked” refers to an arrangement of elements (e.g., a regulatory element such as a promoter) and a coding sequence (nucleic acid sequence ) wherein the elements are positioned such that expression (e.g., transcription) of the joined coding sequence occurs under the direction of the regulatory element. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when it is in the cis position to the coding sequence and the proper transcriptional molecules (e.g., enzymes, transcription factors, etc.) are present.

[0033] In the context of a regulatory element (a promoter), a gene, or the polypeptide product of a gene, “tissue specific” means that the polypeptide product of the gene is detectable in cells of a particular tissue or cell type, but not substantially detectable in certain other cell types. Accordingly, a tissue specific promoter is only active, or its activity is substantially increased, in a specific cell type (e,g, a T cell). Any tissue specific promoter may be used in adenoviral vectors of the disclosure. Tissue specific promoters may be naturally occurring (i.e., obtained from the genome of cells), or they may be synthetic (i.e., designed and constructed using genetic engineering techniques so they function in a specific cell type. A promoter that is specific for a tissue results in a level of expression in that tissue, of a gene operably linked to the tissue-specific promoter, that is at least 2X, at least 5X, or at least 10X, greater than the level of the expression of the gene in a different tissue. In some aspects, the level of expression may be at least 1 log, at least 2 logs, at least 3 logs, at least 4 logs, at least 5 logs, or at least 6 logs greater than the level of gene expression in a non-specific tissue. For example, a T-cell specific promoter is a promoter that results in high level expression of a nucleic acid sequence to which it is operably linked in T-cells, but which does not result in substantially detectable expression of the coding sequence in non- T-cells. In some aspects, the tissue specific promoter may be specific for T-cells.

[0034] One aspect of the disclosure is a system for in vivo CRISPR- mediated production of CAR T-cells, the system comprising:

[0035] a) a first T-cell targeted adenovirus particle that comprises a first adenovirus vector comprising an expression cassette flanked by a 5’ polynucleotide arm and a 3’ polynucleotide arm; wherein the 5’ polynucleotide arm is homologous to a first polynucleotide sequence in a safe harbor locus of a T-cell; wherein the 3’ polynucleotide arm is homologous to a second polynucleotide sequence in the safe harbor locus; and, wherein the expression cassette comprises a first heterologous nucleic acid molecule comprising a tissue-specific promoter operably linked to a nucleic acid sequence encoding a CAR protein; and,

[0036] b) a second T-cell targeted adenovirus particle that comprises a second adenovirus vector comprising a second heterologous nucleic acid molecule, wherein the second heterologous nucleic acid molecule comprises: i) a first mammalian promoter operably linked to a nucleic acid sequence encoding a Cas protein; and, optionally, ii) a second mammalian promoter operably linked to a nucleic acid sequence encoding a gRNA that recognizes a third polynucleotide sequence in the safe harbor locus. In some aspects of the disclosure, the second T-cell targeted adenovirus particle does not comprise a second mammalian promoter or a nucleic acid sequence encoding a gRNA, and the system comprises a third T-cell targeted adenovirus particle that comprises a third adenovirus vector comprising a third heterologous nucleic acid molecule, wherein the third heterologous nucleic acid molecule comprises a second mammalian promoter operably linked to a nucleic acid sequence encoding a gRNA that recognizes a third polynucleotide sequence in the safe harbor locus. In these aspects, the first and second mammalian promoters may, but need not, be a promoter from the same gene (e.g., both may be from the elongation factor alpha (EF~ la) gene). In some aspects, the first polynucleotide sequence is 5’ to the second polynucleotide sequence in the safe harbor locus.

[0037] “CRISPR” and “CRISPER”, may be used interchangeably, and refer to a genetic engineering technique based on the bacterial CAS antiviral defense system that may be used, at least, to edit nucleic acid sequences, including introducing polynucleotides into other nucleic acid molecules. “CRISPER components” refer to molecules necessary to achieve nucleic acid editing using the CRISPR technique, and include, but are not limited to, a CRISPR associated protein (Cas), and a guide RNA (gRNA). Numerous Cas proteins are currently known, and compositions and methods of the disclosure may use any Cas protein suitable for the intended purpose. Examples of Cas proteins suitable for use include, but are not limited to Casl, Cas2, Cas3, Cas8, Cas9, and Casl2. In the CRISPR system, gRNA may comprise two parts: crisprRNA which is the guide sequence that recognizes the target sequence in the genome, and tracrRNA, which the scaffold two which the Cas nuclease binds. The crsprRNA and the tracrRNA may be two separate molecules or they may exist as a single molecule (sgRNA). Thus, as used herein, gRNA encompasses both gRNA in which the crsprRNA and the tracrRNA are two separate molecules, and sgRNA. [0038] “CRISPR/Cas-mediated production of CAR. T-cells” means the use of CRISPR technology to produce T-cells expressing a CAR.. Such production may occur in cells in tissue culture or it may occur in cells within an individual. The terms "individual", "subject", and "patient" are well-recognized in the art and are herein used interchangeably to refer to any animal that produces T-cells. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs. The terms individual, subject, and patient by themselves, do not denote a particular age, sex, race, and the like. Thus, individuals of any age, whether male or female, are intended to be covered by the present disclosure and include, but are not limited to the elderly, adults, children, babies, infants, and toddlers. Likewise, the methods of the present disclosure can be applied to any race, including, for example, Caucasian (white), African- American (black), Native American, Native Hawaiian, Hispanic, Latino, Asian, and European. CRISPR/Cas-mediated production of CAR T-cells in vivo may comprise administering targeted adenovirus particles of the disclosure to an individual so that the CAR-encoding expression cassette is inserted into the genome of a T-cell, resulting in the T- cell expressing the CAR.

[0039] An “expression cassette” refers to a nucleic acid molecule essentially containing a transcriptional regulatory element, such as a promoter, operably linked to a nucleic acid sequence encoding (coding sequence) a protein. Thus, for example, a tissue-specific promotor operably linked to a CAR-encoding nucleic acid sequence may be considered an expression cassette. In some aspects, an expression cassette may contain other regulatory elements that affect transcription, such as response elements, and activator and/or suppressor sequences.

[0040] A “mammalian promoter” means any promoter that is recognized by a mammalian (e.g., human) RNA polymerase and can promote transcription of a gene operably linked thereto in a mammalian cell. Such promoters may, but need not, originate in a mammalian cell. For example, viruses that infect mammalian ceils contain promoters that are recognized by the mammalian transcription apparatus and would thus be considered mammalian promoters. Any mammalian promoter may be used in vectors and methods of the disclosure. In some aspects, a promoter of the disclosure may be a tissue-specific promoter. Examples of suitable mammalian promoters include, but are not limited to, the EF-1α, promoter, a type III RNA polymerase III (U6) promoter, a ubiquitin C (Ubc) promoter, a cytomegalovirus (CMV) promoter, a similar virus 40 (SV40 promoter), a cytokeratinl9 (K 19) promoter, a dLck promoter, a CD3δ promoter.

[0041] A “polynucleotide arm” refers to a polynucleotide molecule that is homologous to a polynucleotide sequence (target sequence) in a safe harbor locus of a cell (e.g., a T-cell) genome. As used herein, “homologous” means that the polynucleotide arm is highly similar (at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 identical) in sequence to the polynucleotide sequence in the safe harbor locus. Methods of the disclosure use polynucleotide arms to facilitate insertion of an expression cassette into the genome of a cell. In some aspects, the polynucleotide arms may flank the expression cassette (hence the name “arms”) which facilitates CRISPR-mediated insertion of the expression cassette into genome of the cell. The length of the polynucleotide arms should be sufficient to allow CRISPR-mediated insertion of the arms, and the flanked expression cassette, into the genome. For example, the polynucleotide arms may be at least 50 nucleotides (nt), at least 100 nt, at least 200 nt, at last 500 nt, at least 750 nt, at least 1000 nt, or at least 1500 nt in length. A “5’ polynucleotide arm” is joined to the 5’ end of the expression cassette, while a “3’ polynucleotide arm” is joined to the 3’ end of the expression cassette.

[0042] The terms “safe harbor locus”, safe harbor gene”, “genomic safe harbor “ may be used interchangeably herein and refer to a location within a genome, including a region of genomic DNA or a specific site, that can be used for integrating an exogenous nucleic acid (e.g., an expression cassette) wherein the integration does not cause any significant deleterious effect on the growth of the host-cell by the addition of the exogenous nucleic acid alone. That is, a safe harbor locus refers to a gene or loci in the genome that a nucleic acid sequence (e.g., expression cassette) can be inserted such that the sequence can integrate and function in a predictable manner (e.g., express a protein of interest) without significant negative consequences to endogenous gene activity, or the promotion of cancer. A safe harbor locus is able to accommodate the integration of new- genetic material in a manner that ensures that the newly inserted genetic elements (i) function predictably and (it) does not cause significant alterations of the host genome thereby averting a risk to the host-cell or organism, and (hi) preferably the Inserted nucleic acid is not perturbed by any read-through expression from neighboring genes, and (iv), does not activate nearby genes. A safe harbor locus may be a specific site or may be a region of the genomic DNA. Examples of safe harbor locus include, but are not limited to, the Rogil locus, the Rogi2 locus, and the Rosa26 locus.

[0043] In some aspects, the first, second, and/or third targeted adenovirus particles may be (Ad5) particles. In some aspects, the first, second, and/or third adenovirus vectors may he Ad5 vectors.

[0044] In some aspects, the 5’ polynucleotide arm is at least 95%, at ieast 96%, at ieast 97%, at least 98%, at least 99%, or 100% identical to the first polynucleotide sequence in the safe harbor locus. In some aspects, the 3’ polynucleotide arm is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the second polynucleotide sequence in the safe harbor locus.

[0045] In some aspects, the tissue-specific promoter may be a T-cell specific promoter, in some aspects, the CAR recognizes a cancer-associated antigen, which may be an ovarian cancer-associated antigen. In some aspects, the cancer associated antigen may be EphA2. In some aspects, the first mammalian promoter is selected from the group consisting of an EF-la promoter, a U6 promoter, a IJ^'be promoter, a CMV promoter, an SV40 promoter, a K19 promoter, a dl.ck promoter, and a CD35 promoter.

[0046] In some aspects, the safe harbor locus is selected from the group consisting of the Rogi 1 locus, the Kogi2 locus, and the Rosa26 locus.

In some aspects, the first, second and/or third targeted adenovirus particles comprise a modified fiber such that the adenovirus particles are targeted to T-cells. In such modified particles, the modified fiber may comprise a heterologous targeting ligand. In some aspects, the modified fiber may comprise a heterologous targeting ligand inserted into the Hi loop of the fiber knob domain. In some aspects, the modified fiber may comprise an Ad5 fiber in which the knob domain has been replaced with the knob domain from the fiber of a serotype 4 adenovirus, which may be an NADC-1 strain of porcine adenovirus type 4, In some aspects, the modified fiber may comprise an Ad 5 fiber in which the amino acid region from about amino acid residue 400 to about amino acid 582 has been replaced with the amino acid region spanning from about amino acid 120 to about amino acid 703 of the knob domain from the fiber of the serotype 4 adenovirus. In some aspects, the modified fiber comprises an RGD domain. In some aspects, the modified fiber comprises a UAO. In some aspects, the targeted adenovirus comprising the modified fiber may have increased infectivity of lymphocytes. In some aspects, a targeted adenovirus comprising the modified fiber may have increased infectivity of T-cells. In some aspects, a targeted adenoviais comprising the modified fiber may have reduced infectivity of ceils that are not T-cells.

[0047] In some aspects, the first, second and/or third targeted adenovirus particles may comprise a modified hexon such that the adenovirus particles have reduced hepatotropism. In some aspects, the modified hexon may comprise an alteration to HVR7. In some aspects, the modified hexon may be produced by replacing HVR7 with a HVR from another serotype of adenovirus. In some aspects, the modified hexon may be produced by replacing HVR7 of Ad5 hexon with HVR7 from adenovirus serotype 3 (Ad3). In some aspects, the modified hexon may comprise an Ad5 hexon in which HVR7 is replaced with HVR7 from adenovirus serotype 3 (Ad3). In some aspects, the modified hexon may comprise an Ad5 hexon in which the amino acid region from about amino acid 382 to about amino acid 588 is replaced with the corresponding region of hexon from Ad3.

[0048] One aspect of the disclosure is use of a system of the disclosure for treating cancer in an individual. Such aspect comprises administering to the individual a first T-cell targeted adenovirus particle that comprises a first adenovirus vector comprising an expression cassette flanked by a 5’ polynucleotide arm and a 3’ polynucleotide arm; wherein the 5’ polynucleotide arm is homologous to a first polynucleotide sequence in a safe harbor locus of a T-cell; wherein the 3’ polynucleotide arm is homologous to a second polynucleotide sequence in the safe harbor locus; wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence in the safe harbor locus; and, wherein the expression cassette comprises a first heterologous nucleic acid molecule comprising a tissue- specific promoter operably linked to a nucleic acid sequence encoding a CAR protein; and,

[0049] b) a second T-cell targeted adenovirus particle that comprises a second adenovirus vector comprising a second heterologous nucleic acid molecule, wherein the second heterologous nucleic acid molecule comprises: i) a first mammalian promoter operably linked to a nucleic acid sequence encoding a Cas protein; and, optionally, ii) a second mammalian promoter operably linked to a nucleic acid sequence encoding a gRNA that recognizes a third polynucleotide sequence in the safe harbor locus. In some aspects of the disclosure, the second T-cell targeted adenovirus particle does not comprise a second mammalian promoter or a nucleic acid sequence encoding a gRNA, and the system comprises a third T-cell targeted adenovirus particle that comprises a third adenovirus vector comprising a third heterologous nucleic acid molecule, wherein the third heterologous nucleic acid molecule comprises a second mammalian promoter operably linked to a nucleic acid sequence encoding a gRNA that recognizes a third polynucleotide sequence in the safe harbor locus. In these aspects, the first and second mammalian promoters may, but need not, be a promoter from the same gene (e.g., both may be from the elongation factor alpha (EF- ia) gene).

[0050] In some aspects, the first, second, and/or third targeted adenovirus particles may be (Ad5) particles. In some aspects, the first second, and/or third adenovirus vectors may be Ad 5 vectors.

[0051] In some aspects, the 5’ polynucleotide arm is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the first polynucleotide sequence in the safe harbor locus. In some aspects, the 3’ polynucleotide arm is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the second polynucleotide sequence in the safe harbor locus.

[0052] In some aspects, the tissue-specific promoter may be a T-cell specific promoter. In some aspects, the CAR recognizes a cancer-associated antigen, which may be an ovarian cancer-associated antigen. In some aspects, the cancer associated antigen may be EphA2. In some aspects, the first mammalian promoter is selected from the group consisting of an EF-la promoter, a U6 promoter, a Ubc promoter, a CMV promoter, an SV40 promoter, a K19 promoter, a dLck promoter, and a CD36 promoter.

[0053] In some aspects, the safe harbor locus is selected from the group consisting of the Rogil locus, the Rogi2 locus, and the Rosa26 locus.

In some aspects, the first, second and/or third targeted adenovirus particles comprise a modified fiber such that the adenovirus particles are targeted to T-cells. In such modified particles, the modified fiber may comprise a heterologous targeting ligand. In some aspects, the modified fiber may comprise a heterologous targeting ligand inserted into the HI loop of the fiber knob domain. In some aspects, the modified fiber may comprise an Ad5 fiber in which the knob domain has been replaced with the knob domain from the fiber of a serotype 4 adenovirus, which may be an NADC-1 strain of porcine adenovirus type 4. In some aspects, the modified fiber may comprise an Ad5 fiber in which the amino acid region from about amino acid residue 400 to about amino acid 582 has been replaced with the amino acid region spanning from about amino acid 120 to about amino acid 703 of the knob domain from the fiber of the serotype 4 adenovirus. In some aspects, the modified fiber comprises an RGD domain. In some aspects, the modified fiber comprises a UAD. In some aspects. the targeted adenovirus comprising the modified fiber may have increased infeetivity of lymphocytes. In some aspects, a targeted adenovirus comprising the modified fiber may have increased infeetivity of T-cells. in some aspects, a targeted adenovirus comprising the modified fiber may have reduced infeetivity of cells that are not T-cells.

[0054] In some aspects, the first, second and/or third targeted adenovirus particles may comprise a modified hexon such that the adenovirus particles have reduced hepatotropism. In some aspects, the modified hexon may comprise an alteration to HVR7. In some aspects, the modified hexon may be produced by replacing HVR7 with a HVR from another serotype of adenovirus. In some aspects, the modified hexon may be produced by replacing HVR7 of Ad5 hexon with HVR7 from adenovirus serotype 3 (Ad3). In some aspects, the modified hexon may comprise an Ad 5 hexon in which HVR7 is replaced with HVR7 from adenovirus serotype 3 (Ad3). in some aspects, the modified hexon may comprise an Ad5 hexon in which the amino acid region from about amino acid 382 to about amino acid 588 is replaced with the corresponding region of hexon from Add.

[0055] in some aspects, the first, second, and optionally third T-cell targeted adenovirus particles may be administered concurrently. Concurrent administration may comprise administeringthe first, second, and optionally third T-cell targeted adenovirus particles by administering a single composition comprising the particles. In some aspects, the first, second, and optionally third T-cel I targeted adenovirus particles may be administered separately. In aspects in which the first, second, and optionally third T-cell targeted adenovirus particles are administered separately, the adenovirus particles may be administered in any order. Moreover, the time between administration of the various adenovirus particles may be immediate, or may be at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 12 hours, or at least 24 hours.

[0056] In aspects of the disclosure in which a targeted adenovirus particle comprise a modified fiber comprising a LOAD, a bridging agent that recognizes the UAD may be administered to the individual.

[0057] Targeted adenovirus particles of the disclosure may be administered to an individual, including human beings, in any suitable formulation and by any suitable route of administration. In some aspects, targeted adenoviral particles may be administered by intravenous (IV) injection, intraperitoneal (IP) injection, intramuscular injection, or by any other parenteral route. Parenteral administration may be performed by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain fomiulatory agents such as suspending, stabilizing and/or dispersing agents.

[0058] To facilitate delivery of the targeted adenovirus particles, the particle may be mixed with a carrier or excipient. Carriers and excipients that might be used include saline, saline buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin). EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. USP grade carriers and excipients are particularly useful for delivery of virions to human subjects. The use of such excipients is well known in the art.

[0059] Targeted adenovirus particles of the disclosure may also be formulated for depot administration. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by IM injection. Thus, for example, die targeted adenovirus particles may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins.

[0060] In some aspects, the cancer may be selected from the group consisting of lung cancer, kidney cancer, brain cancer, colon or rectal cancer, esophageal cancer, breast cancer, ovarian cancer, stomach cancer, liver cancer, prostate cancer, melanoma and leukemias.

[0061] One aspect of the disclosure is a T-cell targeted adenovirus particle that comprises an adenovirus vector comprising an expression cassette flanked by a 5’ polynucleotide arm and a 3’ polynucleotide arm; wherein the 5’ polynucleotide arm is homologous to a first polynucleotide sequence in a safe harbor locus of a T-cell; wherein the 3’ polynucleotide arm is homologous to a second polynucleotide sequence in the safe harbor locus; wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence in the safe harbor locus; and, wherein the expression cassette comprises a heterologous nucleic acid molecule comprising a tissue-specific promoter operably linked to a nucleic acid sequence encoding a CAR protein.

[0062] In some aspects, the targeted adenovirus particle may be an (Ad5) particle. In some aspects, the adenovirus vector may be Ad5 vector. [0063] In some aspects, the 5" polynucleotide arm is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the first polynucleotide sequence in the safe harbor locus. In some aspects, the 3’ polynucleotide arm is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the second polynucleotide sequence in the safe harbor locus.

[0064] In some aspects, the tissue-specific promoter may be a T-cell specific promoter. In some aspects, the CAR recognizes a cancer-associated antigen, which may be an ovarian cancer-associated antigen. In some aspects, the cancer associated antigen may be EphA2. In some aspects, the tissue-specific promoter is selected from the group consisting of a dLck promoter and a CD35 promoter.

[0065] In some aspects, the safe harbor locus is selected from the group consisting of the Rogil locus, the Rogi2 locus, and the Rosa26 locus.

[0066] In some aspects, the first, second and/or third targeted adenovirus particles may comprise a modified fiber such that the adenovirus particles are targeted to T- cells. In such modified particles, the modified fiber may comprise a heterologous targeting ligand. In some aspects, the modified fiber may comprise a heterologous targeting ligand inserted into the H1 loop of the fiber knob domain. In some aspects, the modified fiber may comprise an Ad5 fiber in which the knob domain has been replaced with the knob domain from the fiber of a serotype 4 adenovirus, which may be an NAOC-1 strain of porcine adenovirus type 4. In some aspects, the modified fiber may comprise an Ad5 fiber in which the amino acid region from about amino acid residue 400 to about amino acid 582 has been replaced with the amino acid region spanning from about amino acid 120 to about amino acid 703 of the knob domain from the fiber of the serotype 4 adenovirus. In some aspects, the modified fiber comprises an RGD domain. In some aspects, the modified fiber comprises a UAD, In some aspects, the targeted adenovirus comprising the modified fiber may have increased infectivity of lymphocytes. In some aspects, a targeted adenovirus comprising the modified fiber may have increased infectivity of T-celis. In some aspects, a targeted adenovirus comprising the modified fiber may have reduced infectivity of cells that are not T-celis.

[0067] In some aspects, the first, second and/or third targeted adenovirus particles may comprise a modified hexon such that the adenovirus particles have reduced hepatotropism. In some aspects, the modified hexon may comprise an alteration to HVR7. In some aspects, the modified hexon may be produced by replacing HVR7 with a HVR from another serotype of adenovirus, in some aspects, the modified hexon may be produced by replacing HVR7 of Ad5 hexon with HVR7 from adenovirus serotype 3 (Ad3). In some aspects, the modified hexon may comprise an Ad5 hexon in which HVR7 is replaced with HVR7 from adenovirus serotype 3 (Ad3). In some aspects, the modified hexon may comprise an Ad5 hexon in which the amino acid region from about amino acid 382 to about amino acid 588 is replaced with the corresponding region of hexon from Ad3.

[0068] One aspect of the disclosure is an adenovirus vector comprising an expression cassette flanked by a 5’ polynucleotide arm and a 3’ polynucleotide arm; wherein the 5’ polynucleotide arm is homologous to a first polynucleotide sequence in a safe harbor locus of a T-cell; wherein the 3’ polynucleotide arm is homologous to a second polynucleotide sequence in the safe harbor locus; wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence in the safe harbor locus; and, wherein the expression cassette comprises a heterologous nucleic acid molecule comprising a tissue- specific promoter operably linked to a nucleic acid sequence encoding a CAR protein

[0069] In some aspects, the 5" polynucleotide arm is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the first polynucleotide sequence In the safe harbor locus. In some aspects, the 3’ polynucleotide arm is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the second polynucleotide sequence in the safe harbor locus.

[0070] In some aspects, the tissue-specific promoter may be a T-cell specific promoter. In some aspects, the CAR recognizes a cancer-associated antigen, which may be an ovarian cancer-associated antigen. In some aspects, the cancer associated antigen may be EphA2. In some aspects, the tissue-specific promoter is selected from the group consisting of a dLck promoter and a CD35 promoter.

[0071] In some aspects, the safe harbor locus is selected from the group consisting of the Rogil locus, the Rogi2 locus, and the Rosa26 locus.

[0072] One aspect is a kit comprising a system of the disclosure, or at least one targeted adenovirus particle of the disclosure. Kits may also comprise additional components such as vials, buffers, syringes, and the like. Kits may also comprise instructing administration of a system, or a targeted adenoviral particle of the disclosure, to an individual for treating a disease, such as cancer.

[0073] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

EXAMPLES

[0074] Example 1. In vivo targeting via adenoviral engineering

[0075] This example demonstrates that adenovirus particle and vector engineering can accomplish effective in vivo targeting via vascular administration. A Type 5 adenovirus (Ad5) was modified to express green fluorescent protein (GFP). In addition, the adenovirus particle was targeted to epithelial cell by modifying the Ad fiber knot domain so that it contained myeloid binding protein (MBP)(Ad5.MBP). Of note, vascular epithelium is considered relatively refractory to infection by adenovirus. Either Ad5 expressing GFP and having the intact Ad5 fiber, or Ad5 expressing GFP and the MBP-containing fiber were administered to mice by IV infusion, and at a later time point, the vascular endothelial cells examined by fluorescent analysis. The results are shown in FIG. 3. FIG.3 shows that vascular epithelial cells from mice treated with adenovirus having the intact Ad5 fiber protein exhibit little fluorescence. Vascular epithelial cells from mice treated with adenovirus expressing fiber containing the MBP exhibit significant fluorescence, demonstrating that the presence of the MBP in the fiber allowed those adenoviruses to infect vascular epithelial cells.

[0076] Next, epithelial cells were contacted with either Ad5 containing a GFP under the control of a CMV promoter, or Ad5 containing a GFP gene under the control of a R0B04 promoter, which is specific for epithelial cell expression. After a period of time, the cells were examined for fluorescent expression. As seen in FIG. 3 (bottom), the GFP gene under control of the CMV promoter was not expressed, while the GFP gene under the control of the R0B04 promoter was expressed.

[0077] Example 2. Adenovirus infection of T cells expressing binding receptor

[0078] This example demonstrates that T -cells can be infected by adenovirus If an accessible receptor is expressed on the surface. Murine leukocytes are normally resistant to infection with adenovirus due to deficient expression of the adenovirus receptor. The hCAR +/- mouse model constitutively expresses the adenovirus receptor. The spleens of WT and hCAR mice were harvested and the leukocytes purified. The purified leukocytes were transduced, at various multiplicities of infection (MOIs) with Ad5 expressing GFP under the control of a CMV promoter, and fluorescence measured. The results, which are shown in FIG. 4C. show that hCAR +/- leukocytes, but not wt leukocytes, are susceptible to adenovirus transduction in a dose dependent manner.

[0079] Example 3. Augmented gene transfer using a tropism modified adenovirus

[0080] This example demonstrates that gene transfer to T cell is dramatically enhanced using a tropism modified adenovirus. The spleens of WT and hCAR mice were harvested, the leukocytes purified, and T-cells isolated using magnetic sorting. The purified T-cells were then transduced at various MOIs with one of two different Ad5 expressing GFP under the control of a CMV promoter. In one case, the GFP-expressing Ad5 expressed an intact Ad5 fiber (Ad5). In the second case, the GFP-expressing Ad5 also expressed a fiber in which the knob domain was replaced with the knob region from an NADC-1 strain of porcine adenovirus type 4 adenovirus (Ad5PK4). Fluorescence was then measured using flow cytometry (FIGS. 5C & 5D). The results, which are shown in FIG. 5D, demonstrate that altering the knob region of the fiber resulted in dramatically increased infection of T-cells.

[0081] Example 4. Augmented in vivo gene transfer using a tropism modified adenovirus

[0082] This example demonstrates that gene transfer to T cell is dramatically enhanced using a tropism modified adenovirus. Mice were injected retro- orbitally with either PBS or 5 x 10¹⁰ of Ad5 expressing GFP and either wt Ad5 fiber (Ad5.CMV.eGFP) or a fiber in which the knob domain was replaced with the knob region from an NADC-1 strain of porcine adenoviais type 4 adenovirus (Ad5PK4.CMV.eGFP). After 48 hours, spleens were harvested, and fluorescence measured in T-cells using flow cytometry. The results, which are shown in FIG. 6C, demonstrate that adenoviruses having the knob domain from the porcine adenovirus type 4 fiber had significantly increased infectivity of T-cells.

[0083] Example 5. In vivo gene editing of T lymphocytes using tropism modified adenovirus [0084] This example demonstrates the use of a two-particle system to transfer donor DNA into the genome of a T-cell. Mice were injected i.v. with 5-10 x 1010 either PBS, or 5-10 x 10¹⁰ of: i) an adenovirus containing a donor DNA; and ii) an adenovirus expressing Cas9 and a gRNA. Each adenovirus also expressed a fiber containing RGD-4C peptide, which targets integrins. After 72 hours, spleens were harvested from the mice, T- cells isolated and tested for the presence of the donor DNA by PCR. The results ( FIG. 7B) show that the genomes of T-cells from the mice contain the donor DNA, demonstrating CRISPR-mediated gene transfer via tropism modified adenoviruses.

Claims

WHAT IS CLAIMED IS:

1. A system for CRISPR-mediated production of CAR T cells, comprising: a) a first targeted adenovirus particle that comprises a first adenovirus vector comprising an expression cassette flanked by a 5’ polynucleotide arm and a 3’ polynucleotide arm; wherein the 5’ polynucleotide arm is homologous to a first polynucleotide sequence in a safe harbor locus of a T cell; wherein the 3’ polynucleotide arm is homologous to a second polynucleotide sequence in the safe harbor locus; and, wherein the expression cassette comprises a heterologous nucleic acid molecule comprising a tissue-specific promoter operably linked to a first heterologous nucleic acid sequence encoding a CAR; and, b) a second targeted adenovirus particle that comprises a second adenovirus vector, wherein the second adenovirus vector comprises: i) a second heterologous nucleic acid molecule comprising a first mammalian promoter operably linked to a nucleic acid sequence encoding a Cas protein; and, optionally, ii) a third heterologous nucleic acid molecule comprising a second mammalian promoter operably linked to a nucleic acid sequence encoding gRNA that recognizes a third polynucleotide sequence in the safe harbor locus.

2. The system of claim 1 wherein the second targeted adenovirus particle does not comprise the third heterologous nucleic acid molecule, and wherein the system comprises a third targeted adenovirus particle comprising a third adenovirus vector, wherein the third adenovirus vector comprise a heterologous nucleic acid molecule comprising a second mammalian promoter operably linked to a nucleic acid sequence encoding a gRNA that recognizes a third polynucleotide sequence in the safe harbor locus.

3. The system of claim 1 or 2, wherein each targeted adenovirus particle comprises a modified fiber and/or a modified hexon.

4. The system of claim 3, wherein the T-cell infectivity of the targeted adenovirus particle comprising the modified fiber is greater than the T-cell infectivity of an adenovirus particle having an unmodified fiber.

5. The system of claim 3 or 4, wherein the modified fiber comprises an Ad5 fiber in which the knob domain has been replaced with the knob domain from the fiber of an NADC-1 strain of porcine adenovirus type 4.

6. The system of any one of claims 3-5, wherein the modified hexon has reduced affinity for coagulation factor X.

7. The system of any one of claims 3-6, wherein the modified hexon comprises an Ad5 hexon in which HVR7 has been replaced with HVR7 from adenovirus serotype 3 (Ad3).

8. The system of any one of claims 2-7, wherein the first, second and third adenoviral particles are type 5 adenoviral particles.

9. The system of any one of claims 1-8, wherein the first, second and third adenoviral vectors are 5 adenoviral vectors.

10. The system of any one of claims 1-9, wherein the CAR recognizes a cancer- associated antigen.

11. The system of any one of claims 1-10, wherein the tissue-specific promoter is a T-cell specific promoter.

12. The system of any one of claims 1-11, wherein the safe harbor locus is selected from the group consisting of the Rogi1 locus, the Rogi2 locus, and the Rosa26 locus.

13. Use of the system of any one of claims 1-12 for treating an individual having cancer.

14. An adenovirus vector comprising an expression cassette flanked by a 5’ polynucleotide arm and a 3’ polynucleotide arm; wherein the 5’ polynucleotide arm is homologous to a first polynucleotide sequence in a safe harbor locus of a T cell; wherein the 3’ polynucleotide arm is homologous to a second polynucleotide sequence in the safe harbor locus; and, wherein the expression cassette comprises a heterologous nucleic acid molecule comprising a T-cell-specific promoter operably linked to a heterologous nucleic acid sequence encoding a CAR.

15. A targeted adenovirus particle comprising the adenovirus vector of claim 14, wherein the adenovirus particle is targeted to T cells.