Adeno-associated viral vectors for gene therapy

A virus, amino acid technology, applied in gene therapy, virus/phage, virus, etc., can solve the problem of lack of identifiable molecules, lack of tumor-destroying molecules, etc.

Pending Publication Date: 2020-08-07
INTIMA BIOSCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these successes have been largely limited to hematological tumors, while broader application to solid tumors has been limited by the lack of identifiable molecules expressed by cells in a particular tumor and available to specifically bind tumor targets to mediate Molecules of tumor destruction

Method used

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  • Adeno-associated viral vectors for gene therapy
  • Adeno-associated viral vectors for gene therapy
  • Adeno-associated viral vectors for gene therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0473] Example 1: AAV / CRISPR genome modification of human T cells Day -3: recovery and stimulation

[0474] 10% human serum was added to X-VIVO15 medium and pre-warmed at 37°C. Thaw human PBMCs in a water bath. Immediately after thawing, cells were resuspended and centrifuged at 300 g for 5-10 minutes. Cells were washed with PBS and counted by hemocytometer. Cells were then resuspended in X-VIVO15 + 10% human serum + 300 U / ml IL-2 + 5 ng / ml IL-7 and 11-15 at a concentration of one million cells / ml. Anti-CD3 and anti-CD28 Dynabeads were added and stimulation was performed at a cell:bead ratio of approximately 1 to 2. Cells were cultured for 3 days.

[0475] Day 0: Bead removal, transfection and transduction

[0476] Cells were washed with PBS and placed in DynaMag15 for approximately 1 minute. The beads were washed twice and the pelleted cells were resuspended in X-VIVO15+serum+IL-2+IL-7+IL-15 at a concentration of one million cells / ml. Cells were then incubated at 37°C ...

Embodiment 2

[0485] Example 2: Determination of TCR expression by flow cytometry

[0486] Cells were evaluated by flow cytometry after electroporation with CRISPR at CTLA-4, PD-1, AAVS1 and CISH and on day 3 and beyond (typically days 3, 7 and 14) after transduction with rAAV6 TCR transgene expression. Cell aliquots were collected from cultures, washed, pelleted, and resuspended in Ab (eBioscience Anti-Mouse TCR beta PE) diluted 1 / 100 in PBS + 0.5% FBS - 50-100 ul / sample. Resuspend cells in about 50 to 100 ul of Ab. Cells were incubated at 4°C for 30 minutes. The viability dye eFluor780 or SYTOX Blue viability stain was also added according to the manufacturer's instructions. After incubation, cells were washed twice in PBS and resuspended in 100 to 200 ul of PBS for analysis.

[0487] On days 3, 7 and 14, for AAV6 modified with CRISPR at CTLA-4, PD-1, AAVS1 and CISH and transduced with WT AAV6 or F129L mutated AAV6 (F129L mutated in VP1) and 2 encoding an exogenous TCR cells, their T...

Embodiment 3

[0493] Example 3: Clinical expansion of AAV-modified T cells

[0494] To generate large numbers of transduced T cells, T cell proliferation was induced using a rapid expansion protocol (REP). T cells were initially cultured with anti-CD3, anti-CD28 and IL-2 prior to use in REP and transduced as described above the day after the start of culture. Cells were incubated at 37 °C and 5% CO at 75 cm 2 cultured in flasks. Cells were counted every two days and quantified at 0.5 × 10 6 Cells / mL were suspended in fresh T cell medium containing 300 IU / mL of IL-2 and kept in culture for the rest of the time.

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Abstract

Genetically modified compositions, such as adeno-associated viral vectors and primary cells, for treating various conditions and diseases are disclosed. Disclosed are also modified adeno-associated viruses for the treatment of cancer. Also disclosed are the methods of making and using the genetically modified compositions in treating various diseases, conditions, and cancer.

Description

[0001] cross reference [0002] This application claims US Provisional Application No. 62 / 527,937, filed June 30, 2017, US Provisional Application No. 62 / 659,472, filed April 18, 2018, and US Provisional Application No. 62 / 665,256, filed May 1, 2018 U.S. Provisional Application No. , which is incorporated herein by reference. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on June 29, 2018, is named 47533-726_601_SL.txt and is 5,955,965 bytes in size. Background technique [0005] Despite significant advances in cancer treatment over the past 50 years, there are still many tumor types that are refractory to chemotherapy, radiation therapy, or biological therapy, especially at advanced stages that cannot be resolved by surgical techniques. Recently, significant advances have been made in the genetic engi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17C12N5/0783C12N15/63C12N15/74C12N15/113C12N15/861C12N15/864C07H21/04
CPCC12N15/86C07K14/005C12N9/22C12N2750/14122C12N2750/14143C12N2310/20A61K48/005C07K14/7051C07K2319/00C12N2750/14145A61P35/00A61K35/17A61K48/00
Inventor 托马斯·亨利马维斯·阿格班德耶-麦肯纳蒂尔曼·布尔克斯图默尔莉蒂亚·维尼莫达希尔·乔杜里
Owner INTIMA BIOSCI INC
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