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Liver-specific tropism of adeno-associated viruses

A liver and virus technology, applied in the field of liver tropism control of AAV, can solve problems such as liver tropism

Pending Publication Date: 2021-02-12
MASSACHUSETTS EYE & EAR INFARY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, some AAV serotypes will exhibit liver tropism, which may or may not be desirable depending on the disease being treated

Method used

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  • Liver-specific tropism of adeno-associated viruses
  • Liver-specific tropism of adeno-associated viruses
  • Liver-specific tropism of adeno-associated viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1—Materials and Methods for Characterizing AAV Liver Switching

[0104] By changing the Anc80 scaffold sequence (SEQ ID NO: 1; figure 1 11 positions in ) ( figure 1 X in 1 to X 11 ) to generate 2 of the AAV sequence 11 (2048) - Anc80 library of variants (see, eg, US Patent No. 9,695,220). Each variant was cloned into a mammalian expression plasmid and transfected into HEK293 cells together with the pRep and pAd Helper helper plasmids to generate the library in the form of viral vectors. This library was then used for in vivo screening of liver localization (eg, enrichment vs. off-target). Briefly, in one experiment, three mice were injected with 2.7311 total GC (~1e13 GC / kg) with the Anc80 vector library. On day 3 post-injection, mice were sacrificed, collected to dry and freeze. In another experiment, two rhesus macaques were injected with 1.6e12 gc / kg of Anc80 vector library, the study was terminated and livers were harvested on day 28 after injecti...

Embodiment 2

[0107] Example 2 - Generation and Detection of AAV Liver Switching

[0108] Specific sequences comprising the liver switch were generated from the 2048-variant Anc80-library (see, eg, US Patent No. 9,719,070, the entire contents of which are incorporated herein by reference) (Figure 2). Anc80L65 (SEQ ID NO:2) contained a G at position 266 and showed liver enrichment, and Anc80L65 G266A (SEQ ID NO:3) contained an A at position 266 and showed liver off-target. The G / A liver switch highlighted and color coded in Figure 3 is consistent with the color coding shown in Figure 2.

[0109] Each individual variant can be obtained by site-directed mutagenesis or isothermal (Gibson) cloning in combination with PCR-generated and gene-synthesized fragments. These variants are cloned into standard rep / cap "trans" plasmids to produce vectors. Then, vector variants expressing GFP and α-1-antityrpin were produced at the Gene Transfer Vector Core at the Grousbeck Gene Therapy Center.

[011...

Embodiment 3

[0111] Example 3 - Identification of comparable liver switch residues in other serotypes

[0112] Figure 13A The crystal structure of the AAV2 VP3 capsid monomer is shown. The location of the liver switch ("LT") is indicated by an arrow, and the structure of the region surrounding the LT is indicated by a box. The hepatic switch residues can be located in the 2-3 residues N-terminal to the α-switch in a position comparable to position 266 of Anc80. Figure 13B is a schematic representation of the crystal structure showing coverage of the LT region from AAV 2, 3, 6, 8 and 9VP3, highlighting the local secondary structure defining the switch site. The toggle region is defined as the residues located between and including the secondary structure β-ascending (βa) and β-descending (βd) with 2 to 3 residues encoding the N-terminal to the α-switch (αt) main functional groups. The LT residue is located in VR1, a loop bounded by a β-sheet, N-terminal to a 3-residue α-helix.

[0...

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Abstract

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

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 841,179 filed on April 30, 2019 and U.S. Provisional Application Serial No. 62 / 670,543 filed on May 11, 2018. [0003] field of invention [0004] The present disclosure relates generally to altered tissue tropism of adeno-associated virus (AAV), and specifically to controlling the liver tropism of AAV. Background technique [0005] Adeno-associated virus (AAV) is a parvovirus belonging to the genus Parvovirus, which in turn belongs to the family Parvoviridae. The virus is a replication-deficient, non-enveloped virus that infects humans and some primates but is not known to cause disease. AAV is capable of infecting dividing and quiescent cells and persists in an extrachromosomal state without integrating into the host cell's genome, although in natural viruses, virus-borne genes may also integrate into the host genome. These characteristic...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N7/00C12R1/93
CPCC12N15/86C12N2750/14143C12N2750/14145C12N2750/14122C07K14/005C12N15/102
Inventor L.H.范登伯格P.施密特C.蒂珀C.昂祖E.津恩
Owner MASSACHUSETTS EYE & EAR INFARY
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