Altering tissue tropism of adeno-associated viruses

Patent metadata

Field	Value
Publication number	US-2023051611-A1
Application number	US-201917053412-A
Country	US
Kind code	A1
Filing date	May 10, 2019
Priority date	May 11, 2018
Publication date	Feb 16, 2023
Grant date	—

Abstract

This disclosure provides compositions and methods for altering or changing the tissue tropism, e.g., liver tropism, of adeno-associated viruses (AAV).

First claim

1 . A method of altering tissue tropism of an adeno-associated virus (AAV) vector, the method comprising:
locating an amino acid position within an AAV capsid protein corresponding to position 266 in a capsid protein of Anc80 (SEQ ID NO:1) or corresponding to position 168 in a capsid protein of Anc80 (SEQ ID NO:1); and replacing a naturally occurring amino acid at the located position corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO:1) with a glycine (G) amino acid residue to enhance liver tropism of the resulting AAV vector, or with an alanine (A) amino acid residue to reduce liver tropism of the resulting AAV or replacing a naturally occurring amino acid at the located position corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO:1) with an arginine (R) amino acid residue to enhance liver tropism of the resulting AAV vector, or with a lysine (K) amino acid residue to reduce liver tropism of the resulting AAV.

2 . The method of claim 1 , comprising replacing the naturally occurring amino acid at the located position corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO:1) with a G amino acid residue to enhance liver tropism of the resulting AAV vector.

3 . The method of claim 1 , comprising replacing the naturally occurring amino acid at the located position corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO:1) with an A amino acid residue to reduce liver tropism of the resulting AAV vector.

4 . The method of claim 1 , comprising replacing the naturally occurring amino acid at the located position corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO:1) with an R amino acid residue to enhance liver tropism of the resulting AAV vector.

5 . The method of claim 1 , comprising replacing the naturally occurring amino acid at the located position corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO:1) with a K amino acid residue to reduce liver tropism of the resulting AAV.

6 . The method of claim 1 , wherein the replacing step is performed using site-directed mutagenesis.

7 . The method of claim 1 , wherein the replacing step is performed by restriction digest and ligation of extant or de novo synthesized DNA.

8 . The method of claim 1 , wherein the replacing step is performed by homology-mediated assembly of extant or de novo synthesized DNA.

9 . The method of claim 1 , wherein the locating step is performed by sequencing.

10 . A method of screening a level of liver tropism of an adeno-associated virus (AAV), the method comprising:
sequencing a nucleic acid that encodes for an AAV capsid protein; locating an amino acid position within the AAV capsid protein corresponding to position 266 in a capsid protein of Anc80 (SEQ ID NO:1) or an amino acid position within the AAV capsid protein corresponding to position 168 in a capsid protein of Anc80 (SEQ ID NO:1); and identifying an AAV capsid protein having a G amino acid residue or an A amino acid residue at the located position corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO:1) or having an R amino acid residue or a K amino acid residue at the located position corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO:1), wherein a G amino acid residue at the located position corresponding to position 266 in a capsid protein of Anc80 (SEQ ID NO:1) or an R amino acid residue at the located position corresponding to position 168 in a capsid protein of Anc80 (SEQ ID NO:1) indicates an AAV that exhibits liver tropism, and wherein an A amino acid residue at the located position corresponding to position 266 in a capsid protein of Anc80 (SEQ ID NO:1) or a K amino acid residue at the located position corresponding to position 168 in a capsid protein of Anc80 (SEQ ID NO:1) indicates an AAV that exhibits little or no liver tropism.

11 . An adeno-associated virus (AAV) having the sequence shown in SEQ ID NO:1 [Anc80], wherein X 3 at position 266 is selected from a G or an A or wherein X 1 at position 168 is selected from an R or a K.

12 . An adeno-associated virus (AAV) having a sequence selected from the group consisting of SEQ ID NO: 2 [Anc80L65], SEQ ID NO: 3 [Anc80L65 G266A], SEQ ID NO: 4 [AAV9 G267A], SEQ ID NO: 5 [AAV9 G267A S269T], SEQ ID NO: 6 [AAV9 Anc80L65-VRI], SEQ ID NO: 7 [AAV9 Anc80L65 G266A-VRI], SEQ ID NO: 8 [AAV3B A266G], SEQ ID NO: 9 [AAV3B A266G S267 N268T], SEQ ID NO: 10 [AAV3B G265 A266A], SEQ ID NO: 11 [AAV3B G265 A266G], SEQ ID NO: 12 AAV3B G265 A266A S268T], SEQ ID NO: 13 [AAV3B G265 A266G S268T], SEQ ID NO: 14 [AAV3B AAV9-VRI], SEQ ID NO: 15 [AAV3B Anc80L65-VRI], SEQ ID NO: 16 [AAV3B Anc80L65 G266A-VRI], and SEQ ID NO: 17 [Anc80L65 R168K].

13 . A method of altering tissue tropism of an adeno-associated virus (AAV) vector, the method comprising:
locating a liver toggle region as defined in FIG. 14 within an AAV capsid protein; and replacing a naturally occurring liver toggle region with a liver toggle region from a heterologous serotype or de novo derived sequence to enhance liver tropism or reduce liver tropism of the resulting AAV vector.

14 . The method of claim 13 , wherein, when the heterologous or de novo derived liver toggle region comprises a G amino acid residue at a position within an AAV capsid protein corresponding to position 266 in a capsid protein of Anc80 (SEQ ID NO:1), liver tropism of the resulting AAV vector is enhanced.

15 . The method of claim 13 , wherein, when the heterologous or de novo derived liver toggle region comprises an A amino acid residue at a position within an AAV capsid protein corresponding to position 266 in a capsid protein of Anc80 (SEQ ID NO:1), liver tropism of the resulting AAV vector is reduced.

Assignees

• Massachusetts Eye & Ear Infirmary
• Schepens Eye Res Inst

Inventors

• Vandenberghe Luk H
• Schmit Pauline
• Tipper Christopher
• Unzu Carmen
• Zinn Eric

Classifications

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N2750/14145

  Special targeting system for viral vectors · CPC title

• C07K14/005

  from viruses · CPC title

• C12N2750/14143

  viral genome or elements thereof as genetic vector · CPC title

• C12N2750/14122

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title