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Combinatorial gene therapy

A combination therapy, transgenic technology, applied in the direction of gene therapy, gene therapy composition manufacturing, bone/connective tissue cells, etc., can solve the problem of ineffective transduction of cell types and tissues, virus-mediated transduction elimination, reduction, etc. question

Pending Publication Date: 2021-06-11
EVOX THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] All viral vectors have several disadvantages for gene delivery: (1) vectors of any type cannot efficiently transduce a large number of clinically relevant cell types and tissues, (2) due to prior exposure to wild-type virus, a substantial proportion of patients Pre-existing antibodies to multiple viral vectors (i.e., are seropositive), resulting in abolished or markedly reduced virus-mediated transduction, (3) have been reported against most, if not all, viral vectors, even against AAV, induces humoral, innate, and cellular immune responses (Calcedo et al., Hum Gene Ther Methods, 2018), (4) In diseases where the pathology is complete or partial lack of specific proteins, the expression of transgene products may trigger therapeutic targeting. Proteins themselves or T cell-mediated immune responses against cells transduced with viral vectors (Hollinger & Chamberlain, Curr Opin Neurol, 2018; Sherman et al., Front Immunol, 2017), (5) Virus-mediated specific transgene products Expression is uncontrolled and often unpredictable, which can lead to gene expression levels that are too high, causing toxicity, or too low, causing a lack of therapeutic activity, and finally (6) due to DNA dilution in growing organs, episomal gene expression (e.g. , as a result of AAV-mediated gene therapy) decreases automatically over time, which is a considerable problem especially for pediatric monogenic diseases
However, repeated administration of viral gene therapy remains a considerable challenge for any gene therapy application, especially for genetically ineffective conditions

Method used

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Examples

Experimental program
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preparation example Construction

[0038] In a further aspect, the invention relates to a method of preparing a combination therapy of the invention. The preparation method of the present invention may generally comprise the following steps: (a) infecting a first host cell with a viral vector genome comprising a transgene, and collecting the viral vector produced by said first host cell, and (b) genetically modifying a second host cells to produce EV-based vectors containing the transgene in DNA or RNA polynucleotides (e.g., mRNA, circRNA, minicircle DNA, linear DNA, and / or pDNA polynucleotides), and collected from a second host Cell-produced EV-based vectors. These two steps can be performed simultaneously, sequentially and / or in parallel in any order.

[0039] In preferred embodiments, the first host cell for viral vector production and the second host cell for EV-based vector production are the same cell type. Suitable cell sources include amnion-derived cells; placenta-derived cells; amnion epithelial cel...

Embodiment 1

[0059] Example 1: Repeated delivery of nano-luciferase transgenes using AAV and MSC EVs

[0060] AAV vectors were prepared in HEK293T producer cells by using an adenoviral nanoluciferase expression construct according to standard protocols. Human immortalized MSCs were engineered by lentiviral gene transfer to stably express fusion constructs between CD63 and the NA-binding protein Cas13 or PUF to endogenously encapsulate nanoluciferase mRNA into EVs. EVs were isolated and concentrated using tangential flow filtration (TFF) and bead elution chromatography. Nanoparticle tracking analysis was performed on AAV vectors and isolated EVs to determine particle concentrations. 1e10 AAV particles, or 1e10 EVs, or both 1e10 AAV particles and 1e10 EVs were then added with fresh medium to 6 wells seeded with Huh 7 cells at 50% confluence. Incubate the cells at 37 °C and 5% CO 2 Incubate for 36 hours, then wash twice with PBS. Cells were lysed, substrate added, and luciferase signal qu...

Embodiment 2

[0061] Example 2: Anti-eGFP antibody production is different between AAV and EV-mediated gene therapy

[0062] This example aims to demonstrate that gene therapy using a combination of a viral vector for transgene delivery (in this case AAV9) and an EV-based vector for delivery of the same transgene can be administered repeatedly in vivo without against a second dose of anti-GMO antibody. EV-mediated transgene delivery after AAV9-mediated delivery was compared with the readministration of AAV vectors, in which the AAV vector encoding eGFP and the corresponding mRNA endogenously loaded with eGFP-encoding AAV vector were prepared following the same principle as above. (using CD81-Cas6 fusion protein for mRNA loading), but here both vector types were produced in HEK293T host cells. After (i) administration of AAV9eGFP vector (1e10 AAV9 particles) once ( image 3 AAV in ), (ii) after repeated administration of AAV9eGFP vector ( image 3 AAV+AAV in AAV), and (iii) an in vitro an...

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Abstract

The present invention relates to administration of gene therapy, in particular the development of effective combinatorial strategies for gene delivery in inter alia monogenetic diseases. More specifically, the present invention relates to a combination therapy, methods of producing the combination therapy, as well as various related embodiments.

Description

technical field [0001] The present invention relates to the administration of gene therapy, in particular to the development of effective combinatorial strategies for gene delivery, especially in monogenic diseases. Background technique [0002] Gene therapy aims to correct defective genes that underlie disease development. A common approach to this problem involves delivering normal genes to the nucleus. This gene can then be inserted into the genome of the target cell or can remain episomal. Delivery of the corrective gene to target cells of a subject can be accomplished by a variety of methods, including the use of viral vectors. In recent years, various recombinant gene transfer vectors based on different types of viruses (eg, retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, etc.) have been developed and tested in clinical trials. Adeno-associated virus (AAV)-based gene transfer vectors have become favored vectors due to properties such as the abili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00
CPCA61K48/0025A61K48/0083C12N2750/14143Y02A50/30C12N5/0662C12N9/0069C12N15/86C12N2509/00A61K48/0091
Inventor S·埃尔安达劳西P·伦丁
Owner EVOX THERAPEUTICS LTD