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Novel recombinant adeno-associated virus capsids with enhanced human pancreatic tropism

A capsid and virus technology, applied in the direction of viruses, viral peptides, viruses/bacteriophages, etc., can solve complex problems

Active Publication Date: 2020-11-27
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, pancreatic gene therapy trials require high levels of transduction because there are physical limitations on how much AAV can be delivered in a single intraperitoneal injection due to the need to ensure that the injected vector specifically and efficiently targets the patient. is compounded by the fact that diseased pancreatic tissue or islet cells

Method used

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  • Novel recombinant adeno-associated virus capsids with enhanced human pancreatic tropism
  • Novel recombinant adeno-associated virus capsids with enhanced human pancreatic tropism
  • Novel recombinant adeno-associated virus capsids with enhanced human pancreatic tropism

Examples

Experimental program
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Effect test

example 1

[0381] Example 1: Directed evolution of AAV for human islet targeting

[0382] Introduction

[0383]Adeno-associated virus (AAV) has attracted great interest as a potent vehicle for gene transfer into various cell types. Although AAV has several features that make itself a promising vehicle for human gene therapy, several disadvantages hinder the use of AAV in clinical applications, such as its promiscuous nature, limited transgene package size, and pre-existing neutralizing antibodies in the general population high prevalence. For gene therapy purposes, transduction needs to be both efficient and highly cell-type specific. AAV cell tropism and immunogenicity are determined by the sequences of the structural capsid proteins VP1, VP2 and VP3.

[0384] DNA shuffling is a powerful method for the evolution of molecules with specific functions in vitro and has applications in various fields such as medicine, pharmacy and agricultural research. Shuffling of AAV capsid sequences ...

example 2

[0391] Example 2: Novel recombinant adeno-associated virus capsids with enhanced human pancreatic tissue or human islet cell tropism

[0392] Purpose

[0393] The purpose of the present invention is to find rAAV vectors with enhanced ability to transduce human islet cells. This could be useful for new treatments targeting endocrine disorders, especially type 1 and type 2 diabetes.

[0394] Technical Description

[0395] AAV capsid libraries were used to select for AAVs that selectively transduce dissociated or intact human islets. These capsid sequences were amplified using barcode-specific reverse primers and used to generate rAAV with GFP as the transgene ( Figure 18 ). Islets dissociated using Accumax were transduced at MOI = 1K or 10K μg / cell for 48 hours at 37°C (10% FBS / CMRL), plated 48 hours after transduction and analyzed by flow cytometry. Intact human islets were transduced on ice at MOI=10K μg / cell for 1-2 hours (2% FBS / CMRL), followed by suspension culture fo...

example 3

[0404] Example 3: Generation and screening of a barcoded adeno-associated virus capsid shuffling library for enhanced transduction of primary human islet cells

[0405] Summary

[0406] Safe and efficient gene transfer to Langerhans islets is a promising approach for treating diabetes. Recombinant AAV-mediated gene transfer into islets has been hampered by the lack of AAV serotypes that efficiently transduce these cells. To identify novel AAV serotypes with improved tropism for human islets, two highly complex, barcoded, replication-competent capsid libraries were constructed, diversity verified by single-molecule DNA sequencing, and performed on human islets sequence selection. The enriched barcodes were tracked by high-throughput sequencing, and three capsid variants were identified that were able to generate 5-fold to 10-fold better capsids than the best previously identified capsids. Highly efficient transduction of dissociated and intact islets. These novel AAV capsid...

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Abstract

The present invention relates to variant AAV capsid polypeptides, wherein the variant AAV capsid polypeptides exhibit increased transduction and / or tropism in human pancreatic tissue or human islets as compared non-variant parent capsid polypeptides.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 651,010, filed March 30, 2018, and U.S. Provisional Application No. 62 / 745,226, filed October 12, 2018, both of which were adopted by References are hereby incorporated in their entirety. [0003] Statement of Invention Rights in Federally Sponsored Research or Development [0004] This invention was made with US Government support under Grant No. U01DK089569. The US Government has certain rights in this invention. technical field [0005] The present invention relates to a variant AAV capsid polypeptide, wherein the variant AAV capsid polypeptide exhibits increased transduction and / or tropism in human pancreatic tissue or human islets compared to a non-variant parental capsid polypeptide. Background technique [0006] Inherited disorders caused by the absence or defect of a desirable gene (loss of function) or the expression of an undesirabl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/005C12N15/86C12N15/10
CPCC07K14/005C12N15/1027C12N15/1058C12N15/86C12N2750/14122C12N2750/14143C12N2750/14151A61K47/6901C07K14/075C12N7/00C12N15/102C12N15/902C12N2710/10041C12N2710/10071C12N2710/10134C12N2710/10141C07K14/015C12N2750/14123
Inventor 卡特娅·佩克伦马克·A·凯
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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