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Engineered viral vector reduces induction of inflammatory and immune responses

a technology of inflammatory and immune responses, applied in the field of recombinant viruses, can solve the problems of compromising the immune system of patients during treatment, hammering the therapeutic effect of humans, etc., and achieve the effects of reducing the immunogenicity of a modified virus, reducing the inflammatory response, and inhibiting the tlr9-mediated inflammation

Inactive Publication Date: 2019-10-17
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The approach effectively diminishes inflammatory responses and increases transgene expression by avoiding TLR9-mediated inflammation, allowing for safer and more effective gene therapy with reduced immune system compromise.

Problems solved by technology

Furthermore, many AAV gene therapy clinical trials do not utilize immunosuppression prophylactically and only administer corticosteroids upon signs of inflammation or tissue damage, which has been associated with variable therapeutic efficacy [22, 23].
Overall, these observations are compatible with immune-mediated destruction of AAV-transduced hepatocytes and demonstrate that inflammatory and immune responses triggered by AAV administration are a safety concern and can hamper therapeutic efficacy in humans.
These immunosuppressive drugs strongly hamper T cell activation and therefore compromise the patient's immune system during treatment.

Method used

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  • Engineered viral vector reduces induction of inflammatory and immune responses
  • Engineered viral vector reduces induction of inflammatory and immune responses
  • Engineered viral vector reduces induction of inflammatory and immune responses

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Modified Viral Genome

[0111]To engineer an AAV vector that has the ability to specifically evade TLR9 activation in immune cells, we inserted two copies of c41 separated by a 5-nucleotide-long spacer (AAAAA; SEQ ID NO: 8) into the 3′ untranslated region of AAV vector encoding enhanced green fluorescent protein (eGFP) (FIG. 3B). Subsequently, we produced wild-type AAV-eGFP virus and AAV-eGFP-c41 virus (harboring two c41 insertions). Infectious titers of both viruses were comparable (˜109 infectious units / ml, as determined by titering on HeLa cells), suggesting that addition of c41 into the viral genome did not hamper viral packaging and infectivity, an important consideration for viral vectors that have to be mass-produced for gene therapy.

example 2

Modified Viral Genome Reduces NF-kB Activation

[0112]To measure the inflammatory response, we used HEK293 cells stably expressing TLR9 (HEK293 TLR9 cells), which senses AAV DNA genomes, and also expressing alkaline phosphatase under the transcriptional control of NF-kB. When NF-kB is activated, which indicates inflammation, alkaline phosphatase is secreted into the media and acts on a provided substrate, leading to a change in color of the media that can be measured on a plate reader. We mock-infected HEK293 TLR9 cells or infected them with either AAV-eGFP or AAV-eGFP-c41. In agreement with the literature, AAV-eGFP infection induced a small but statistically significant increase in NF-kB activity (FIG. 4). In contrast, AAV-eGFP-c41 infection was not significantly different compared to mock-infected cells, indicating that the virus was able to evade eliciting an inflammatory response.

example 3

Modified Viral Genome Transduced More Cells and Expresses More Transgene

[0113]We analyzed the above three conditions (described in FIG. 4) for eGFP expression using flow cytometry. We found that AAV-eGFP-c41 transduced more cells than AAV-eGFP (52.7% GFP+ compared to 34.6% GFP+) (FIG. 5). In addition, GFP+ cells from AAV-eGFP-c41 infection expressed ˜twice as much eGFP as GFP+ cells from AAV-eGFP infection (mean fluorescence intensity [MFI] of 5335 compared to 2749).

[0114]In summary, we engineered an AAV vector to evade TLR9-mediated inflammation by incorporating an inhibitory oligonucleotide in the viral genome.

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Abstract

Modified viral genomes are able to reduce induction of inflammatory and immune anti-viral responses. This manifests itself in reduced NF-kB activity, increased viral transduction rates, and increased expression of transgenes. Viral genomes are modified by incorporating one or more oligonucleotide sequences which are able to bind to TLR9 but not induce activation of it. The oligonucleotide sequences may be synthetic, bacterial, human, or from any other source.

Description

RELATED APPLICATION DATA[0001]This application is continuation of PCT / US2017 / 036525 filed Jun. 8, 2017, which claims priority to U.S. Provisional Application No. 62 / 347,302 filed on Jun. 8, 2016, each of which is herein incorporated by reference in its entirety for all purposes.FEDERALLY SPONSORED RESEARCH[0002]This invention was made with government support under Grant No. HG008525 awarded by National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD OF THE INVENTION[0003]This invention is related to the area of viral based therapies. In particular, it relates to recombinant viruses.BACKGROUND OF THE INVENTION[0004]Gene therapy has immense potential to prevent, treat, and cure multiple human diseases [1]. In 2012, Glybera™ (alipogene tiparvovec) became the first viral gene therapy to be approved for use in the western world [2]. Glybera™ utilizes adeno-associated virus (AAV) as a viral vector to deliver the human lipoprotein lipase (LPL) gene ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/86A61K35/76
CPCC12N15/86C12N2750/00032A61K35/76C12N2750/00041C12N7/00C12N15/117A61K31/7088A61K45/06A61K48/00A61P29/00C12N2750/14143C12N2750/14171C12N2750/14151C12N2330/51C12N2310/17Y02A50/30
Inventor CHAN, YING KAICHURCH, GEORGE
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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