Constructs and Methods for Delivering Molecules via Viral Vectors with Blunted Innate Immune Responses

a technology of innate immune responses and constructs, which is applied in the direction of viruses/bacteriophages, biocide, genetic material ingredients, etc., can solve the problems of recombinant aav, attendant immune responses that have compromised the outcome of aav-mediated gene therapy, and the inability to integrate into the genome, so as to improve adeno-associated virus (aav)-mediated gene expression

Inactive Publication Date: 2014-09-18
THE TRUSTEES OF THE UNIV OF PENNSYLVANIA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]In a further aspect, the invention provides a method for improving adeno-associated virus (AAV)-mediated gene expression by generating an AAV viral particle comprising a modified packaging insert, wherein said packaging insert comprises a nucleic acid molecule comprising AAV inverted terminal repeats (ITRs) sequences or functional equivalents thereof, e.g., 5′ AAV ITR and 3′ AAV ITR (which ITRs may be independently selected from CpG-modified or wild-type ITRs and optionally CpG-modified self-complementary ITRs) and an exogenous gene sequence under the control of regulatory sequences which control expression of the gene product, wherein said sequences of said nucleic acid molecule are modified to reduce CpG di-nucleotides such that an immune response to the vector is reduced as compared to the unmodified AAV vector without significant reduction in expression of the gene product; and delivering the AAV to a subject intramuscularly.

Problems solved by technology

However, recombinant AAV, which does not contain any viral genes and only a therapeutic / marker expression cassette packaged in an AAV capsid, does not integrate into the genome.
However, in some experimental settings, attendant immune responses have compromised the outcome of AAV-mediated gene therapy.

Method used

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  • Constructs and Methods for Delivering Molecules via Viral Vectors with Blunted Innate Immune Responses
  • Constructs and Methods for Delivering Molecules via Viral Vectors with Blunted Innate Immune Responses
  • Constructs and Methods for Delivering Molecules via Viral Vectors with Blunted Innate Immune Responses

Examples

Experimental program
Comparison scheme
Effect test

example 1

In the Absence of TLR9 Signaling, AAVrh32.33nLacZ Muscle Gene Transfer Results in Stable Transgene Expression

[0135]The current study assessed the requirement for TLR9 signaling in T cell immunoreactivity and transgene loss in response to AAVrh32.33. These mechanistic findings were subsequently translated into a modified, CpG-depleted AAVrh32.33 vector that escapes the adaptive immune response and exhibits stable, long-term transgene expression.

[0136]A. Material and Methods:

[0137]1. Mice: C57BL / 6 wild type (WT) mice were ordered from The Jackson Laboratory. Toll-like receptor 9 knockout (TLR9KO) mice were a kind gift from Dr. Phillip Scott (University of Pennsylvania, Philadelphia, Pa.). All mice were housed under specific pathogen-free conditions in the TRL Animal Facility at the University of Pennsylvania. All animal procedure protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

[0138]2. Adeno-Associated Viral Vectors (AAV) me...

example 2

[0177]To measure LacZ expression of RhCpG+ and CpG− constructs, HeLa cells were transfected with CpG+ and CpG− AAV expression plasmids. Four days post transfection cells were assayed for β-galactosidase activity using the Mammalian β-galactosidase assay kit as instructed for adherent cells. Absorbance was measured at 405 nm on a TECAN Infinite M1000 PRO plate reader. CpG+ and CpG− AAV vector constructs exhibited comparable LacZ plasmid expression. These data demonstrate that transgene loss in the skeletal muscle of RhCpG+ gene transferred is not due to differential β-gal expression levels at the plasmid level.

[0178]To assess transgene stability and cellular infiltrate at an early kinetic time point, mice were injected intramuscularly with 1×1011 GC of RhCpG+ or RhCpG− vector. 14 days post vector injection, gastrocnemius was harvested and skeletal muscle cryosections were stained with CD4 or CD8 monoclonal antibody (MAb) as well as X-gal. Stable transgene expression and minimal cellu...

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Abstract

A CpG-modified recombinant adeno-associated viral (AAV) vector is described. The vector carries a nucleic acid molecule comprising AAV inverted terminal repeat (ITR) sequences and an exogenous gene sequence under the control of regulatory sequences which control expression of the gene product, in which the nucleic acid sequences carried by the vector are modified to significantly reduce CpG di-nucleotides such that an immune response to the vector is reduced as compared to the unmodified AAV vector. Also provided are methods and regimens for delivering transgenes using these AAV viral vectors, in which the innate immune response to the vector and / or transgene is significantly modulated.

Description

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0001]This invention was made with government support by 5-T32-HL-007954-12 awarded by National Heart, Lung, and Blood Institute (NHLBI) and 5-T32-AI-007324-20 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0002]Adeno-associated virus (AAV) is a small, non-enveloped human parvovirus that packages a linear strand of single stranded DNA genome that is 4.7 kb. The capsid of an AAV contains 60 copies (in total) of three viral proteins (VPs), VP1, VP2, and VP3, in a predicted ratio of 1:1:10, arranged with T=1 icosahedral symmetry [H-J Nam, et al., J Virol., 81(22): 12260-12271 (November 2007)]. The three VPs are translated from the same mRNA, with VP1 containing a unique N-terminal domain in addition to the entire VP2 sequence at its C-terminal region [Nam et al., cited above]. VP2 contains an extra N-terminal sequence in addition to VP3 at its C te...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/86
CPCC12N15/86C12N2750/14141C07H21/04A61K48/0058A61K48/0075A61K48/0091C12N7/00C12N2750/14142C12N2750/14143C12N2800/24
Inventor RABINOWITZ, JOSEPH E.WILSON, JAMES M.FAUST, SUSAN M.
Owner THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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