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Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs

A gene modification, blood cell technology, applied in the field of immunology, can solve the problem of inactivation of retroviral particles

Pending Publication Date: 2021-12-24
埃克苏马生物技术公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such methods require large amounts of vectors and said methods carry the risk of inactivation of retroviral particles due to coagulation factors and / or other enzymes present in the body
Ultimately, such methods have the risk of high transduction levels of non-target cells / organs

Method used

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  • Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs
  • Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs
  • Methods and compositions for genetically modifying lymphocytes in blood or in enriched pbmcs

Examples

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example 1

[0568] Example 1. Materials and methods for transduction experiments

[0569] This example provides the materials and methods used in the experiments disclosed in the subsequent examples herein.

[0570] Recombinant lentiviral particles are produced by transient transfection.

[0571] Unless otherwise stated, 293T cells (Lenti-X TM 293T, Clontech) suitable for passing in Freestyle TM Suspension cultures (referred to as F1XT cells) were performed with continuous growth in 293 expression medium (ThermoFisher Scientific) and used as packaging cells in the experiments herein.

[0572] It should be noted that a typical 4-vector packaging system includes 3 packaging plastids encoding (i) gag / pol, (ii) rev, and (iii) a pseudotyping element such as VSV-G. The fourth vector of this packaging system is a genomic plastid, a third-generation lentiviral expression vector encoding one or more related genes (with a deletion in the 3'LTR that causes self-inactivation). For transfections u...

example 2

[0580] Example 2. Exposure to retroviral particles pseudotyped VSV-G or influenza HA and NA for 4 hours and optionally with an envelope derived from VSV-G, MV or MuLV and furthermore optionally anti-CD3 on its surface Transduction efficiency of scFv co-pseudotyped unstimulated PBMCs.

[0581] In this example, lentiviral particles pseudotyped or co-pseudotyped by various envelope proteins and optionally displaying T cell activation elements were exposed to unstimulated human PBMC for 4 hours and transduction efficiency assessed .

[0582] Production of recombinant lentiviral particles in F1XT cells. Cells were transiently transfected with PEI with genomic plastids and separate packaging and envelope plastids encoding gag / pol, rev. For some samples, the transfection reaction mixture also included plastids encoding UCHT1 scFvFc-GPI, a co-pseudotyped envelope, or a co-pseudotyped envelope fused to anti-CD3 scFv. In this example, the genomic plastid used for the sample was F1-0-...

example 3

[0587] Example 3. Efficient genetic modification of resting lymphocytes by exposing whole blood to recombinant retroviral particles for 4 hours, followed by a PBMC enrichment procedure.

[0588]In this example, unstimulated human T cells and NKT cells were efficiently genetically modified by a 4-hour incubation of a reaction mixture comprising whole blood and retroviral particles pseudotyped by VSV-G and Displays T cell activation elements on its surface. Next, PBMCs were isolated from the transduction reaction mixture using a conventional density gradient centrifugation-based PBMC enrichment procedure. Transduction of CD3+ cells was assessed by expression of the eTag transgene using flow cytometry.

[0589] Purification using depth filtration The following lentiviral particles were used in this example: F1-3-23 (F1-3-23G) pseudotyped by VSV-G; and F1-3-23G pseudotyped by VSV-G and displaying T cell activation elements F1-3-23 of UCHT1-scFvFc-GPI (F1-3-23GU).

[0590] 10 ml...

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Abstract

The present disclosure provides methods and compositions for genetically modifying lymphocytes, for example T cells and / or NK cells, in shorter times than previously and / or in whole blood or a component thereof. In some embodiments, a lymphodepletion filter assembly is used before or after forming a reaction mixture where lymphocytes are contacted with recombinant retroviral particles in a closed system, to genetically modify the lymphocytes.

Description

[0001] Cross References to Related Applications [0002] This application is a continuation-in-part of International Application No. PCT / US2018 / 051392, filed September 17, 2018; and claims the following rights: U.S. Provisional Application No. 62 / 726,293, filed September 2, 2018 Nos.; U.S. Provisional Application No. 62 / 726,294, filed September 2, 2018; U.S. Provisional Application No. 62 / 728,056, filed September 6, 2018; U.S. Provisional Application No. 62 / 728,056, filed September 17, 2018 62 / 732,528; U.S. Provisional Application No. 62 / 821,434, filed March 20, 2019; and U.S. Provisional Application No. 62 / 894,853, filed September 1, 2019; and International Application No. PCT / US2018 / 051392 is a continuation-in-part of International Application No. PCT / US2018 / 020818, filed March 3, 2018; and claims the following rights: U.S. Provisional Application No. 62 / , filed September 18, 2017 560,176; U.S. Provisional Application No. 62 / 564,253, filed September 27, 2017; U.S. Provision...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N15/867
CPCA01K2207/12A01K2227/105A01K2267/0331C07K14/7051C07K2319/03C12N5/0636C12N5/0646C12N15/86C12N2510/00C12N2740/16043C12N2310/141C12N15/113C12N2320/12A61K2039/80C07K14/70517C07K14/70521C07K2319/00C07K2319/02C07K2319/33C07K2319/43C12N2501/65A61K39/4613A61K39/4631A61K39/464411A61K2239/38A61K39/461A61K39/4611A61K39/464402C07K14/4702C07K16/2809C07K2317/622C07K2319/035C12N7/00C12N15/11C12N2501/91C12N2740/15022
Inventor 格雷戈里·伊恩·弗罗斯特詹姆斯·约瑟夫·奥努弗法扎德·哈里扎德弗雷德里克·维甘特阿尼邦·昆都
Owner 埃克苏马生物技术公司
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