Adeno-associated virus variant capsids and use for inhibiting angiogenesis

A virus, variant technology, applied in the direction of virus/phage, virus, application, etc., can solve problems such as deeper cell types that have not yet been reported

Pending Publication Date: 2020-10-13
4D MOLECULAR THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these AAVs, including AAV2, have not been reported to efficiently transduce deeper cell types of the retina when delivered by intravitreal administration

Method used

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  • Adeno-associated virus variant capsids and use for inhibiting angiogenesis
  • Adeno-associated virus variant capsids and use for inhibiting angiogenesis
  • Adeno-associated virus variant capsids and use for inhibiting angiogenesis

Examples

Experimental program
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Effect test

Embodiment 1

[0343] Intravitreal injection and tissue harvest. Single male cynomolgus monkeys (macaca fascicularis) aged 4 to 10 years and weighing at least 4 kg were administered by intravitreal injection through the sclera (approximately 3 mm posterior to the limbus using procedures and delivery devices adapted for human use). Animals were anesthetized and local anesthetic was administered. 100 μL of library was administered to each eye.

[0344] On day 14±3, euthanasia was performed by trained veterinary personnel using 100 mg / kg sodium pentobarbital intravenously. Eyes were nucleated and stored at 4 °C until dissection.

[0345] tissue dissection. Cut the eye along the serradium with a scalpel and remove the anterior segment. A decompression cut is made on the retina around the fovea to allow a flat mount of the retina and the vitreous is removed. Collect six retinal samples from each quadrant (superior, inferior, nasal, and temporal) as figure 2 Cellular material corresponding ...

Embodiment 2

[0367] Directed evolution was used to discover novel adeno-associated virus (AAV) variants with superior gene delivery to retinal cells following intravitreal (IVT) administration, which is significantly superior to other methods of gene delivery to the human eye The route of administration (Example 1). Cell tropism after intravitreal administration of a novel AAV variant (LAISDQTKHA+P34A; SEQ ID NO:42) comprising a P34A substitution and insertion of the peptide LAISDQTKHA (SEQ ID NO:28) at amino acid 588, in nonhuman primates Representative example of the ability of an AAV variant containing ISDQTKH (SEQ ID NO: 14) to transduce retinal cells assessed in vivo in (NHP).

[0368] Standard methods were used to produce recombinant AAV virions comprising the AAV2 capsid or the novel variant capsid LAISDQTKHA+P34A (SEQ ID NO: 42) and the CMV promoter (AAV2.CMV.GFP and LAISDQTKHA+P34A.CMV, respectively). Genome of a green fluorescent protein (GFP) transgene operably linked to the CA...

Embodiment 3

[0372] Cell tropism of a novel AAV variant LAISDQTKHA+P34A (SEQ ID NO:42) for retinal pigment epithelial (RPE) and photoreceptor (PR) cells, induced in vitro using RPE cells and from fibroblast-derived human PR cells derived from competent stem cells (FB-iPSCs) or human embryonic stem cells (ESCs) were evaluated.

[0373]Standard methods were used to produce AAV virions comprising an AAV2 capsid or a novel variant capsid LAISDQTKHA+P34A (SEQ ID NO:42) and comprising a CAG promoter (AAV2.CAG.EGFP and LAISDQTKHA+P34A.CAG, respectively). .EGFP) Genome of an operably linked green fluorescent protein (EGFP) transgene. Human RPE cell cultures were generated from the human embryonic stem cell line ESI-017 or human fibroblast-derived induced pluripotent stem cells ("FB-iPSCs") using a 45-day differentiation protocol. Maturation into RPE cells was confirmed by measuring: the expression of mature RPE markers including RPE65 and BEST1; the synthesis of VEGF and PEDF; and the ability to ...

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Abstract

Provided herein are variant adeno-associated virus (AAV) capsid proteins having one or more modifications in amino acid sequence relative to a parental AAV capsid protein, which, when present in an AAV virion, confer increased infectivity of one or more types of retinal cells as compared to the infectivity of the retinal cells by an AAV virion comprising the unmodified parental AAV capsid protein.Also provided are recombinant AAV virions and pharmaceutical compositions thereof comprising a variant AAV capsid protein as described herein, methods of making these rAAV capsid proteins and virions, and methods for using these rAAV capsid proteins and virions in research and in clinical practice, for example in, e.g., the delivery of nucleic acid sequences to one or more cells of the retina forthe treatment of retinal disorders and diseases.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application Serial Nos. 62 / 590,976, filed November 27, 2017, and 62 / 664,726, filed April 30, 2018, the entire disclosures of each of which are incorporated herein by reference. [0003] Incorporation by reference of the sequence listing provided as a text file [0004] The sequence listing is provided herein as a text file "090400-5010-WO-seq-listing.txt" created on November 26, 2018 and having a size of 238KB. The contents of the text file are hereby incorporated by reference in their entirety. technical field [0005] The invention disclosed herein relates generally to the field of adeno-associated virus (AAV) virions comprising variant capsid proteins and the use of directed evolution techniques to generate such variant capsids. Background technique [0006] Inherited retinal diseases encompass a large heterogeneous group of genetic disorders that a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/13C07K14/015A61K39/395A61K9/00A61P27/02A61P27/06A61P9/10A61P35/00
CPCA61K9/0048A61K48/0008A61K48/005A61K48/0075C07K16/22C07K14/005C12N15/86C12N2750/14122C12N2750/14143C12N2750/14145C12N7/00
Inventor D.柯恩M.科特曼D.谢弗P.什曼斯基P.弗兰西斯
Owner 4D MOLECULAR THERAPEUTICS INC
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