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KR-20260065692-A - Adeno-associated virus variant and use thereof

KR20260065692AKR 20260065692 AKR20260065692 AKR 20260065692AKR-20260065692-A

Abstract

An adeno-associated virus (AAV) capsid protein variant with improved infectivity to target cells and transduction efficiency, and its use are disclosed. Specifically, an AAV2 variant capsid protein selected through directed evolution for gene delivery to retinal cells, a recombinant AAV vector containing the same, and its use as a gene delivery vehicle to retinal cells are disclosed.

Inventors

  • 장재형
  • 이준원
  • 김유진
  • 정한

Assignees

  • 연세대학교 산학협력단

Dates

Publication Date
20260511
Application Date
20241101

Claims (15)

  1. AAV2 variant capsid protein comprising H38P, I698V, N705A, V708A, T716N, V719L, S721T, and N734P point mutations at positions corresponding to 38, 698, 705, 708, 716, 719, 721, and 734 of the wild-type adeno-associated virus (AAV)2 capsid protein of SEQ ID NO. 1.
  2. The AAV2 variant capsid protein of claim 1, wherein the AAV2 variant capsid protein is composed of an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO. 2.
  3. The AAV2 variant capsid protein of claim 1, wherein the AAV2 variant capsid protein is an AAV2 variant capsid protein that confers increased infectivity to retinal cells compared to the wild-type AAV2 capsid protein.
  4. AAV2 variant capsid protein according to claim 3, wherein the retinal cells are selected from the group consisting of retinal ganglion cells, bipolar cells, photoreceptor cells, Müller glial cells, horizontal cells, amacrine cells, astrocytes, microglia, retinal vascular cells, and retinal pigment epithelial cells.
  5. Isolated nucleic acid encoding the AAV2 variant capsid protein of claim 1.
  6. A host cell comprising isolated nucleic acid encoding the AAV2 variant capsid protein of claim 1.
  7. AAV2 variant capsid proteins comprising H38P, I698V, N705A, V708A, T716N, V719L, S721T, and N734P point mutations at positions corresponding to 38, 698, 705, 708, 716, 719, 721, and 734 of the wild-type AAV2 capsid protein of SEQ ID NO. 1, and A recombinant AAV vector comprising a heterogeneous nucleic acid containing a sequence encoding a target product.
  8. Claim 7, wherein the recombinant AAV vector is a recombinant AAV vector that results in increased transduction of retinal cells compared to an AAV vector containing wild-type AAV2 capsid protein.
  9. A recombinant AAV vector according to claim 7, wherein the object product is a polypeptide or nucleic acid.
  10. A pharmaceutical composition for treating ocular diseases comprising the recombinant AAV vector of claim 7.
  11. A pharmaceutical composition according to claim 10, wherein the ocular disease is selected from retinal-vitreous-choroidal related disease, retinal vascular disease, macular disease, hereditary retinal disease, hereditary vitreous disease, hereditary choroidal disease, intraocular inflammatory disease, intraocular tumor, glaucoma, and optic neuropathy.
  12. A pharmaceutical composition according to claim 10, wherein the eye disease is selected from macular degeneration, diabetic retinopathy, retinal vascular occlusion, macular telangiectasia, retinopathy of prematurity, myopic degeneration, retinitis pigmentosa, Leber congenital amaurosis, Best's disease, Stargardt's disease, congenital non-progressive night blindness, X-linked retinal detachment, Vietti's crystal retinopathy, total color blindness, cone cell dystrophy, cone-rod cell dystrophy, macular dystrophy, Usher syndrome, Bardett-Beadle syndrome, syndromic retinitis pigmentosa, panchoroidal atrophy, centrochoroidal dystrophy, cerebral choroidal atrophy, glaucoma, optic neuropathy, uveitis, uveal melanoma, intraocular lymphoma, retinoblastoma, retinal detachment, and retinal injury.
  13. A pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is administered by intravitreal injection, subretinal injection, or macula injection.
  14. An in vitro method for delivering heterogeneous nucleic acids to retinal cells, comprising the step of contacting the recombinant AAV vector of claim 7 with retinal cells.
  15. The method of claim 14, wherein the retinal cells are selected from the group consisting of retinal ganglion cells, bipolar cells, photoreceptor cells, Müller glial cells, horizontal cells, amacrine cells, astrocytes, microglia, retinal vascular cells, and retinal pigment epithelial cells.

Description

Adeno-associated virus variant and use thereof An adeno-associated virus (AAV) capsid protein variant with improved infectivity to target cells and transduction efficiency, and its use are disclosed. Specifically, an AAV2 variant capsid protein selected through directed evolution for gene delivery to retinal cells, a recombinant AAV vector containing the same, and its use as a gene delivery vehicle to retinal cells are disclosed. This research was conducted with support from the Samsung Future Technology Development Program (Project No.: SRFC-MA1901-09). Gene therapy is a method of treating hereditary diseases by introducing normal genes into human cells to correct or compensate for conditions caused by genetic defects and abnormalities, and it has become a key means of treating various hereditary diseases. Adeno-associated viruses (AAVs) are widely used gene delivery vectors in the field of gene therapy because they can infect a wide range of tissues, are non-pathogenic, and have low immunogenicity. It is known that there are various serotypes of AAV, and that the host or viral characteristics differ depending on the serotype. For example, AAV of serotype 2 (AAV2) can infect various cells, and serotypes 1 (AAV1), serotype 5 (AAV5), and serotype 6 (AAV6) have tissue infection specificity compared to AAV2, so AAV1 is known to have high gene delivery efficiency to muscles, liver, airways, central nervous system, etc., AAV5 to the central nervous system, liver, retina, etc., and AAV6 to the heart, muscles, liver, etc. However, to be used as a gene delivery vector for gene therapy, it must possess higher tissue specificity and higher gene delivery and expression efficiency compared to current AAV vectors. AAV capsid engineering is being utilized to develop AAV vectors that meet these requirements. Directed evolution is a widely used high-throughput screening method for engineering improved biomolecules. It mimics the process of natural selection through repeated induction/introduction of genetic mutations and selection. Directed evolution of AAV capsids involves introducing mutations into wild-type AAV capsid genes to generate a library of AAV capsid variants with diverse sequences, and then screening for capsid variants with desired characteristics to identify new capsid variants. Recombinant AAV vectors with high tissue specificity and gene delivery efficiency have been developed and utilized through AAV capsid engineering (Korean Registered Patent 2234930). In particular, hereditary retinal diseases are often monogenetic diseases and are considered promising indications for gene therapy, and AAV variants such as AAV2-7m8, AAV2.GL, and AAV2.NN have been developed. However, there is still a need for AAV variants with excellent transduction capabilities that can be delivered with high specificity to desired cells in the retina to induce gene expression. Accordingly, the inventors completed the present invention by selecting an AAV2 variant with excellent gene transfer efficacy to retinal cells using human retinal organoids from an AAV library constructed by directed evolution. FIG. 1 shows a schematic diagram of a directed evolution method for engineering an AAV2 variant capsid protein according to one embodiment of the present invention. A capsid gene ( cap ) variant was generated using error-prone PCR on the gene encoding the wild-type AAV2 capsid protein, packaged to construct an AAV library, and transduced into human retinal organoids to select AAVs having tropism toward retinal cells. Subsequently, tdTomato+ photoreceptor cells were selected from the human retinal organoids, the cap gene was obtained therefrom, analyzed, and packaged to construct a photoreceptor-oriented AAV library, and the process of transduction, selection, and analysis was repeated to select an AAV2 variant capsid protein with improved transduction toward retinal cells compared to an AAV having a wild-type AAV2 capsid. FIG. 2 illustrates the three-dimensional structure of an RO3 variant according to one embodiment of the present invention, the location of the point mutation relative to the wild-type AAV2 capsid protein is indicated, and an enlarged illustration of the outer and inner surfaces of the region with the point mutation is shown on the right. FIG. 3 illustrates the structure of a recombinant AAV2 variant vector containing an RO3 variant according to one embodiment of the present invention. Figure 4 shows the expression pattern of the target gene observed after subretinal administration of a recombinant AAV2 vector loaded with a gene encoding GFP (green fluorescent protein) as the target gene on an RO3 variant or wild-type AAV2 according to one embodiment of the present invention. FIG. 5 shows the expression pattern of the target gene observed after intravitreal administration of a recombinant AAV2 vector (RO3-GFP vs. AAV2-GFP) loaded with GFP as the target gene on an RO3 variant or wild-type AAV2 according to one embodiment o