# US 2023/0407255 A1 · Dendritic cell assay for innate immunogenicity to gene therapy agents

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- Number: US 2023/0407255 A1
- Document ID: US20230407255A1
- Status: Published application
- Kind: A1
- Application number: 18/299,656
- Filed: 2023-04-12
- Published: 2023-12-21
- Earliest claimed: 2022-04-12
- Assignees: Genzyme Corporation
- Applicants: Genzyme Corporation
- Inventors: Sourav Roy CHOUDHURY; Mona MOTWANI
- Web page: https://patentgrep.com/patent/US20230407255A1

## CPC

- C12N 5/0645: {Macrophages, e.g. Kuepfer cells in the liver; Monocytes}
- G01N 33/6863: {Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C12N 15/86: Viral vectors
- G01N 33/5047: {Cells of the immune system}
- C12N 2502/1121: Dendritic cells
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- G01N 2500/00: Screening for compounds of potential therapeutic value
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Provided herein are methods for determining the innate immunogenicity of a gene therapy agent in an individual. In particular, the methods involve the use of isolated dendritic cells to detect innate immunogenicity to a gene therapy agent. Exemplary gene therapy agents include adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, Herpes simplex virus (HSV) vectors or a lipid nanoparticles.

## Claims

### Claim 1

A method of determining the innate immunogenicity to a gene therapy agent in an individual, the method comprising
a) incubating an innate immune cell from the individual with the gene therapy agent,
b) analyzing the innate immune cell for altered expression of one or more cytokines compared to a suitable control, whereby altered expression of the one or more cytokines produces a cytokine signature,
wherein expression of a cytokine signature following incubation with the gene therapy agent indicates innate immunogenicity to the gene therapy agent in the individual.

### Claim 2 (depends on 1)

The method of claim 1, wherein the innate immune cell is a dendritic cell, a monocyte, a macrophage or a natural killer (NK) cell.

### Claim 3 (depends on 1, 2)

The method of claim 1 or 2, wherein the innate immune cell is isolated from peripheral blood mononuclear cells from the individual.

### Claim 4 (depends on 1, 2, 3)

The method of any one of claims 1-3, wherein the innate immune cell is a dendritic cell.

### Claim 5 (depends on 4)

The method of claim 4, wherein the dendritic cell is derived from a monocyte of the individual.

### Claim 6 (depends on 4, 5)

The method of claim 4 or 5, further comprising isolating monocytes from the individual and incubating the monocytes in dendritic cell culture media to derive dendritic cells from the monocytes prior to incubating the dendritic cells with the gene therapy agent.

### Claim 7 (depends on 6)

The method of claim 6, wherein the monocytes are CD14+ monocytes.

### Claim 8 (depends on 6, 7)

The method of claim 6 or 7, wherein the monocytes are incubated with the dendritic cell culture media for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The method of any one of claims 1-8, wherein the innate immune cells are replated prior to the incubation with the gene therapy agent of step b).

### Claim 10 (depends on 9)

The method of claim 9, wherein the innate immune cells are replated into microwell dishes.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The method of any one of claims 1-10 wherein the gene therapy agent is a viral vector or a non-viral vector.

### Claim 12 (depends on 11)

The method of claim 11 wherein the viral vector is an AAV.

### Claim 13 (depends on 11)

The method of claim 11 wherein the non-viral vector is a lipid nanoparticle (LNP).

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The method of any one of claims 1-10, wherein the gene therapy agent is a viral vector and wherein the innate immune cells are incubated with the gene therapy agent at an MOI of about 1×103 to about 1×105 or about 1×104.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The method of any one of claims 1-10, wherein the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng/mL to about 1 mg/mL.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-15, wherein the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The method of any one of claims 1-16, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

The method of any one of claims 1-17, wherein the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1 and MIP-1α.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of any one of claims 1-18, wherein the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1 and MIP-1α.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of any one of claims 1-18, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The method of any one of claims 1-16, wherein the cytokine signature comprises increased expression of one or more of IP10, MIP1b, CXCL9, and IL2.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The method of any one of claims 1-16, wherein the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, and IL2.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The method of any one of claims 1-22, wherein expression of the cytokines in the cytokine signature is increased compared to a suitable control.

### Claim 24 (depends on 23)

The method of claim 23, wherein the suitable control is the expression of the cytokines in the cytokine signature from innate immune cells that are not incubated with the gene therapy agent or wherein the suitable control is expression of the cytokines in the cytokine signature from innate immune cells prior to incubation with the gene therapy agent.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24)

The method of any one of claims 1-13 and 16-24, wherein the gene therapy agent is a viral vector.

### Claim 26

A method of determining the innate immunogenicity to a viral gene therapy agent in an individual, the method comprising
a) incubating monocytes from the individual in dendritic cell culture media under conditions in which the monocytes differentiate into dendritic cells,
b) incubating the dendritic cells with the viral gene therapy agent at an MOI of about 1×103 to about 1×105 for about 12 to about 36 hours,
c) analyzing the dendritic cells for altered expression of one or more cytokines compared to a suitable control, whereby altered expression of the one or more cytokines produces a cytokine signature,
wherein expression of the cytokine signature following incubation with the viral gene therapy agent indicates innate immunogenicity to the viral gene therapy agent in the individual, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

### Claim 27 (depends on 26)

The method of claim 26, wherein the monocytes are obtained from peripheral mononuclear cells from the individual.

### Claim 28 (depends on 26, 27)

The method of claim 26 or 27, wherein the monocytes are CD14+ monocytes.

### Claim 29 (depends on 26, 27, 28)

The method of any one of claims 26-28, wherein the monocytes are incubated in dendritic cell culture media for about 7-8 days to differentiate the monocytes to dendritic cells.

### Claim 30 (depends on 26, 27, 28, 29)

The method of any one of claims 26-29, wherein the dendritic cells are incubated with the viral gene therapy agent at an MOI of about 1×104

### Claim 31 (depends on 26, 27, 28, 29, 30)

The method of any one of claims 26-30, wherein the dendritic cells are incubated with the viral gene therapy agent for about 24 hours

### Claim 32 (depends on 25, 26, 27, 28, 29, 30, 31)

The method of any one of claims 25-31, wherein the viral vector is an AAV particle.

### Claim 33 (depends on 32)

The method of claim 32, wherein the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, An AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LKO3 capsid, an AAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2 chimeric capsid, a bovine AAV capsid, a mouse AAV capsid rAAV2/HBoV1 (chimeric AAV/human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.B capsid or an AAVPHP.eB capsid, or a functional variant thereof.

### Claim 34 (depends on 33)

The method of claim 33, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

### Claim 35 (depends on 32, 33, 34)

The method of any one of claims 32-34, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs is an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, or an AAV12 ITR.

### Claim 36 (depends on 35)

The method of claim 35, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

### Claim 37 (depends on 35)

The method of claim 35, wherein the one or more ITRs and the capsid of the AAV particles are derived from different AAV serotypes.

### Claim 38 (depends on 25, 26, 27, 28, 29, 30, 31)

The method of any one of claims 25-31, wherein the viral vector is an adenoviral particle.

### Claim 39 (depends on 38)

The method of claim 38, wherein the adenoviral particle comprises an capsid from Adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, or a functional variant thereof.

### Claim 40 (depends on 25, 26, 27, 28, 29, 30, 31)

The method of any one of claims 25-31, where the viral vector is a lentiviral particle.

### Claim 41 (depends on 40)

The method of claim 40, wherein the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross river virus (RRV), Ebola virus, Marburg virus, Mokala virus, Rabies virus, RD114, or a functional variant thereof.

### Claim 42 (depends on 25, 26, 27, 28, 29, 30, 31)

The method of any one of claims 25-31, where the viral vector is a Herpes simplex virus (HSV) particle.

### Claim 43 (depends on 42)

The method of claim 42, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle, or a functional variant thereof.

### Claim 44 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20)

The method of any one of claims 1-10 and 15-20, wherein the gene therapy agent is a lipid nanoparticle.

### Claim 45 (depends on 21, 22)

The method of claim 21 or claim 22, wherein the gene therapy agent is a lipid nanoparticle.

### Claim 46

A method of determining the innate immunogenicity to a non-viral gene therapy agent in an individual, the method comprising
a) incubating monocytes from the individual in dendritic cell culture media under conditions in which the monocytes differentiate into dendritic cells,
b) incubating the dendritic cells with the non-viral vector at a concentration of about 1 ng/mL to about 1 mg/mL,
c) analyzing the dendritic cells for altered expression of one or more cytokines compared to a suitable control, whereby altered expression of the one or more cytokines produces a cytokine signature,
wherein expression of the cytokine signature following incubation with the non-viral gene therapy agent indicates innate immunogenicity to the non-viral gene therapy agent in the individual, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

### Claim 47 (depends on 46)

The method of claim 46, wherein the monocytes are obtained from peripheral mononuclear cells from the individual.

### Claim 48 (depends on 46, 47)

The method of claim 46 or 47, wherein the monocytes are CD14+ monocytes.

### Claim 49 (depends on 46, 47, 48)

The method of any one of claims 46-48, wherein the monocytes are incubated in dendritic cell culture media for about 7-8 days to differentiate the monocytes to dendritic cells.

### Claim 50 (depends on 46, 47, 48, 49)

The method of any one of claims 46-49, wherein the dendritic cells are incubated with the non-viral gene therapy agent for about 12 hours to about 36 hours or about 24 hours

### Claim 51 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)

The method of any one of claims 1-50, wherein the gene therapy agent comprises nucleic acid encoding a heterologous transgene.

### Claim 52 (depends on 51)

The method of claim 51, wherein the heterologous transgene is operably linked to a promoter.

### Claim 53 (depends on 52)

The method of claim 52, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

### Claim 54

A method of determining the innate immunogenicity to a lipid nanoparticle (LNP) in an individual, the method comprising
a) incubating monocytes from the individual in dendritic cell culture media under conditions in which the monocytes differentiate into dendritic cells,
b) incubating the dendritic cells with the LNP,
c) analyzing the dendritic cells for altered expression of one or more cytokines compared to a suitable control, whereby altered expression of the one or more cytokines produces a cytokine signature,
wherein expression of the cytokine signature following incubation with the LNP indicates innate immunogenicity to the non-viral gene therapy agent in the individual.

### Claim 55 (depends on 54)

The method of claim 54, wherein the cytokine signature comprises increased expression of one or more of IP10, MIP1b, CXCL9, and IL2.

### Claim 56 (depends on 54)

The method of claim 54, wherein the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, and IL2.

### Claim 57

A method of determining a cytokine signature of a gene therapy agent comprising
a) incubating one or more innate immune cell from one or more individuals with the gene therapy agent,
b) analyzing the one or more innate immune cells for altered expression of one or more cytokines compared to a suitable control,
wherein the altered expression of one or more cytokines in step b) indicates the cytokine signature of the gene therapy agent.

### Claim 58 (depends on 57)

The method of claim 57, wherein the innate immune cell is a dendritic cell, a monocyte, a macrophage or a natural killer (NK) cell.

### Claim 59 (depends on 57, 58)

The method of claim 57 or 58, wherein the one or more innate immune cells are isolated from peripheral blood mononuclear cells from the individual.

### Claim 60 (depends on 57, 58, 59)

The method of any one of claims 57-59, wherein the innate immune cell is a dendritic cell.

### Claim 61 (depends on 60)

The method of claim 60, wherein the dendritic cell is derived from a monocyte of the one or more individuals.

### Claim 62 (depends on 60, 61)

The method of claim 60 or 61, further comprising isolating monocytes from the one or more individuals and incubating the monocytes in dendritic cell culture media to derive dendritic cells from the monocytes prior to incubating the dendritic cells with the gene therapy agent.

### Claim 63 (depends on 62)

The method of claim 62 wherein the monocytes are CD14+ monocytes.

### Claim 64 (depends on 63)

The method of claim 63 or 63, wherein the monocytes are incubated with the dendritic cell culture media for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes.

### Claim 65 (depends on 57, 58, 59, 60, 61, 62, 63, 64)

The method of any one of claims 57-64, wherein the innate immune cells are replated prior to the incubation with the gene therapy agent of step b).

### Claim 66 (depends on 57, 58, 59, 60, 61, 62, 63, 64, 65)

The method of any one of claims 57-65, wherein the gene therapy agent is a viral vector and wherein the innate immune cells are incubated with the gene therapy agent at an MOI of about 1×103 to about 1×105 or about 1×104.

### Claim 67 (depends on 57, 58, 59, 60, 61, 62, 63, 64, 65)

The method of any one of claims 57-65, wherein the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng/mL to about 1 mg/mL.

### Claim 68 (depends on 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67)

The method of any one of claims 57-67, wherein the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

### Claim 69 (depends on 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68)

The method of any one of claims 57-68, wherein expression of the cytokines in the cytokine signature is increased compared to a suitable control.

### Claim 70 (depends on 69)

The method of claim 69, wherein the suitable control is the expression of the cytokines in the cytokine signature from innate immune cells that are not incubated with the gene therapy agent or wherein the suitable control is expression of the cytokines in the cytokine signature from innate immune cells prior to incubation with the gene therapy agent.

### Claim 71 (depends on 57, 58, 59, 60, 61, 62, 63, 64, 65, 68, 69, 70)

The method of any one of claims 57-65 and 68-70, wherein the gene therapy agent is a viral vector.

### Claim 72 (depends on 66, 71)

The method of claim 66 or 71, wherein the viral vector is an AAV particle.

### Claim 73 (depends on 72)

The method of claim 72, wherein the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, An AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LKO3 capsid, an AAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2 chimeric capsid, a bovine AAV capsid, a mouse AAV capsid rAAV2/HBoV1 (chimeric AAV/human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.B capsid or an AAVPHP.eB capsid, or a functional variant thereof.

### Claim 74 (depends on 73)

The method of claim 73, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

### Claim 75 (depends on 72, 73, 74)

The method of any one of claims 72-74, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs is an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, or an AAV12 ITR.

### Claim 76 (depends on 75)

The method of claim 75, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

### Claim 77 (depends on 75)

The method of claim 75, wherein the one or more ITRs and the capsid of the AAV particles are derived from different AAV serotypes.

### Claim 78 (depends on 66, 71)

The method of claim 66 or 71, wherein the viral vector is an adenoviral particle.

### Claim 79 (depends on 78)

The method of claim 78, wherein the adenoviral particle comprises an capsid from Adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, or a functional variant thereof.

### Claim 80 (depends on 66, 71)

The method of claim 66 or 71, where the viral vector is a lentiviral particle.

### Claim 81 (depends on 80)

The method of claim 80, wherein the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross river virus (RRV), Ebola virus, Marburg virus, Mokala virus, Rabies virus, RD114, or a functional variant thereof.

### Claim 82 (depends on 66, 71)

The method of claim 66 or 71, where the viral vector is a Herpes simplex virus (HSV) particle.

### Claim 83 (depends on 82)

The method of claim 82, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle, or a functional variant thereof.

### Claim 84 (depends on 57, 58, 59, 60, 61, 62, 63, 64, 65, 67, 68, 69, 70)

The method of any one of claims 57-65 and 67-70, wherein the gene therapy agent is a lipid nanoparticle.

### Claim 85 (depends on 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84)

The method of any one of claims 67-84, wherein the gene therapy agent comprises nucleic acid encoding a heterologous transgene.

### Claim 86 (depends on 85)

The method of claim 85, wherein the heterologous transgene is operably linked to a promoter.

### Claim 87 (depends on 86)

The method of claim 86, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

### Claim 88 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87)

A kit for use in the methods of any one of claims 1-87.