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Methods of predicting ancestral virus sequences and uses thereof

A sequence, ancestral technology, applied in the direction of viruses, viral peptides, viruses/phages, etc., can solve problems such as reducing efficiency and increasing safety risks by gene transfer

Pending Publication Date: 2016-12-14
MASSACHUSETTS EYE & EAR INFARY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the subject is already naturally infected with the virus, subsequent treatment with a vector based on the virus results in increased safety risks and reduced efficiency of gene transfer due to cellular and humoral immune responses

Method used

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  • Methods of predicting ancestral virus sequences and uses thereof
  • Methods of predicting ancestral virus sequences and uses thereof
  • Methods of predicting ancestral virus sequences and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1: In silico prediction of ancestral sequences

[0094] A set of 75 different amino acid sequences for AAV capsids were obtained from a number of public databases, including GenBank, and the sequences were aligned using the PRANK-MSA algorithm, version 121002, with option "-F".

[0095] ProtTest3 (see e.g. Darriba et al., 2011, Bioinformatics, 27(8):1164-5; available on the World Wide Web at darwin.uvigo.es / software / prottest3) was used under different conditions (e.g. in ProTest3 Those conditions included, i.e. "+I", "+F", "+G" and their combinations) evaluate different models of peptide evolution (e.g. those included in ProTest3, i.e. JTT, LG, WAG, VT, CpRev, RtRev, Dayhoff, DCMut, FLU, Blosum62, VT, HIVb, MtArt, MtMam). The JTT model was selected based on the Aikake Information Criterion (AIC; Hirotugu, 1974, IEEE Transactions on Automatic Control, 19:716-23) score as implemented in ProTest3 (Jones et al., 1992, Comp. Appl. Biosci., 8: 275-82) and +G and +F ...

Embodiment 2

[0107] Example 2: Expression of Ancestral AAV VP1 Sequences

[0108] Experiments were performed to determine whether the predicted ancestral AAV capsid sequences could be used to make viral vectors.

[0109] A number of predicted ancestral AAV capsid sequences were cloned. Transfer of the ancestral capsid library to the rep-cap expression plasmid enables viral particle formation in a transient transfection. To maintain proper expression levels and splicing of VP1, VP2 and VP3, by cleaving HindIII located 5' of cap in the rep coding sequence and SpeI engineered between the cap stop codon and polyadenylation signal, The library cap genes were cloned. Therefore, to clone the ancestral capsid into the more conventional "REP / CAP" construct, the passage plasmid was digested with HindIII and SpeI, gel purified, and ligated into a similarly digested rep / cap plasmid.

[0110] The expressed polypeptides were resolved on 10% SDS gels. Such as Figure 6 As shown in , capsid polypepti...

Embodiment 3

[0111] Example 3: Virus titration

[0112] AAV was produced in HEK293 cells by transient co-transfection of plasmids encoding all elements required for virion assembly. Briefly, HEK293 cells were grown to 90% confluency and transfected with (a) a viral genome plasmid encoding a luciferase transgene (expressed through a CMV promoter) flanked by AAV2 ITRs, (b) encoding AAV packaging plasmids for AAV2rep and synthetic capsid proteins disclosed herein, (c) AAV2-AAP expression capsid, and (d) adenoviral helper genes required for AAV packaging and assembly. Cells were cultured at 37°C for 2 days, and cells and media were harvested.

[0113] The cell culture medium suspension was lysed by 3 consecutive freeze-thaw cycles. Afterwards, the lysate was clarified by centrifugation and treated with an enzyme (herein Benzonase TM ) to digest any DNA present outside the virus particle. Dilute the AAV prep to fall within the linear measurement range of the control DNA template, in this ca...

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Abstract

Methods are described for predicting ancestral sequences for viruses or portions thereof. Also described are predicted ancestral sequences for adeno-associated virus (AAV) capsid polypeptides. The disclosure also provides methods of gene transfer and methods of vaccinating subjects by administering a target antigen operably linked to the AAV capsid polypeptides.

Description

field of invention [0001] In general, the present disclosure relates to viruses. Background of the invention [0002] Evasion and avoidance of neutralizing or toxic immune responses against gene therapy vectors is a major challenge for all gene transfer vector types. Gene transfer to date is most efficiently achieved using vectors based on viruses circulating in humans and animals, such as adenoviruses and adeno-associated viruses (AAV). However, if the subject has been naturally infected with the virus, subsequent treatment with the virus-based vectors leads to increased safety risks and reduced efficiency of gene transfer due to cellular and humoral immune responses. Capsid antigens are primarily responsible for innate and / or adaptive immunity against viral particles, however, polypeptides encoded by viral genes can also be immunogenic. Summary of the invention [0003] The present disclosure describes methods of predicting and synthesizing ancestral viral sequences, o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/015C12N15/35C12N7/01C12N15/864
CPCC07K14/005C12N7/00C12N15/86C12N2750/14171C12N2750/14143C12N2750/14141C12N2750/14121C12N2750/14122A61K39/00A61P27/02A61P37/04A61P43/00C07K14/075Y02A90/10C07K14/00A61K2039/525A61K2039/6075C12N2750/14142C07K16/22C07K2317/24C07K2317/76C12N2750/14123
Inventor L.H.范登伯格E.津恩
Owner MASSACHUSETTS EYE & EAR INFARY
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