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Adeno-associated-virus rep sequences, vectors and viruses

a technology of vectors and viruses, applied in the field of adeno-associated virus (aav) replication (rep) sequences, can solve the problems of limited success in the prior art strategy of constructing hybrid viruses (e.g., ad/aav) by controlling rep expression, and achieve the effect of high level of rep protein expression

Inactive Publication Date: 2014-06-05
THE RES FOUND OF STATE UNIV OF NEW YORK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method for reducing the level of a molecule, cell, virus, or phenomenon in a sample. This can be done by comparing the level in a sample to a control and calculating the difference. The method can also be used to compare the level in different subjects or at different times. The text also describes the use of the AAV Rep78 protein in a method for SSI (Simultaneous Acquisition of Infection) and the importance of this protein in the process. Overall, the patent text provides a way to measure and compare the levels of molecules, cells, viruses, or phenomena in a sample.

Problems solved by technology

To date, prior art strategies to construct hybrid viruses (e.g., Ad / AAV) by controlling Rep expression have met with limited success.

Method used

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  • Adeno-associated-virus rep sequences, vectors and viruses
  • Adeno-associated-virus rep sequences, vectors and viruses
  • Adeno-associated-virus rep sequences, vectors and viruses

Examples

Experimental program
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Effect test

example 1

Materials and Methods

De Novo Synthesized Constructs:

[0253]All de novo synthesized sequences were synthesized and cloned into pUC13 by GenScript USA. Scrambled Rep, Deoptimized Rep, were designed using a program previously described (13). The sequences were designed with unique restriction sites SbfI and SwaI flanking them as well as a unique R.E AfeI at bp 661 of the AAV2 genome to allow ease of manipulation. A naturally occurring R.E BstBI site that occurs at bp 1623 of the AAV2 genome was retained.

[0254]Rep78 Design I, II, III and IV was designed using algorithms described in the art (Coleman et al., Science 320:1784 (2008). S (wt3) Rep contains wild type Rep sequences from AAV2 bp 1623 to 2186. S (wt1,2) Rep contains scrambled Rep sequences from AAV2 bp 1623 to 2186. The constructs shown in FIG. 3 delimit the 135 bp interval from bp 1782 to bp 1916 of the AAV2 genome.

Plasmids and Cloning:

[0255]For detection of expression levels from wild-type, scrambled and Deoptimized Rep ORFs, ...

example 2

Modification of Rep ORF

[0267]To construct a first generation Ad carrying Rep78, the inventors expressed Rep under a tightly regulated tetracycline inducible promoter within an ΔE1ΔE1 F5 / 35 Adenovirus. The fiber knob of this Ad5 was replaced with that of Ad35 to allow it to infect hematopoietic cells (3). The tetracycline inducible Rep78 expression cassette, has been previously used successfully for the construction of a helper dependent Adenovirus carrying Rep78 (9). Surprisingly, the inventors found that the same construct on an E1 deleted backbone was incapable of replication, showing no signs of viral growth in spite of multiple passages in HEK 293 packaging cells. The inventors hypothesized that the replicative functions provided by multiple helper virus genomes in trans to the helper dependent virus allowed replication, whereas a single genome carrying both Adenoviral genes and the Rep expression construct was unable to escape Rep's inhibitory effect.

[0268]To elucidate the rela...

example 3

Modification of Rep ORF Allows Replication of Adenovirus

[0271]The Scrambled and Deoptimized Rep constructs were cloned downstream of the tetracycline inducible promoter, in place of the wild-type Rep ORF, within the fiber modified first generation Adenovirus genome, generating infectious clones pAd / sRep78 and pAd / dRep78 (FIG. 2A). These viral constructs were linearized and transfected into HEK 293 packaging cells and passaged every 10 days onto fresh cells, until the development of CPE was observed. As mentioned earlier, no signs of viral replication could be observed with pAd / WTRep78 even with passaging up to 50 days. However, complete CPE was observed with both pAd / sRep78 and pAd / dRep78 within a total of 15 days from transfection. Production of both viruses could be scaled up with infectious virus yields comparable to each other and to Ad / AAVFVIII (Table 1), proving a clear role for the sequence of Rep in the inhibition of Adenoviral replication.

TABLE IViral titersVirusTiter† (PFU...

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Abstract

The invention provides adeno-associated virus (AAV) replication (Rep) sequences. In one embodiment, the invention provides nucleotide sequences encoding a chimeric protein, wherein the encoded chimeric protein contains a wild type AAV Rep inhibitory amino acid sequence, and wherein the nucleotide sequences contain a scrambled and / or deoptimized polynucleotide sequence encoding the wild type AAV Rep inhibitory amino acid sequence. The invention provides vectors, cells, and viruses containing the invention's sequences. Also provided are methods for detecting portions of the AAV Rep inhibitory amino acid sequence, which reduce replication and / or infection and / or productive infection by viruses. The invention's compositions and methods are useful for site-specific integration and / or expression of heterologous sequences by recombinant adeno-associated virus (rAAV) vectors and by rAAV virus particles, such as hybrid viruses (e.g., Ad-AAV) comprising such vectors. The invention's compositions and methods find application in, for example, gene therapy and / or vaccines.

Description

[0001]This application claims priority under 35 U.S.C. §119(e) to co-pending U.S. Provisional Application Ser. No. 61 / 476,858, filed on Apr. 19, 2011, herein incorporated by reference in its entirety.[0002]This invention was made with government support under grant AI41636, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.FIELD OF INVENTION[0003]The invention provides adeno-associated virus (AAV) replication (Rep) sequences. In one embodiment, the invention provides nucleotide sequences encoding a chimeric protein, wherein the encoded chimeric protein contains a wild type AAV Rep inhibitory amino acid sequence, and wherein the nucleotide sequences contain a scrambled and / or deoptimized polynucleotide sequence encoding the wild type AAV Rep inhibitory amino acid sequence. The invention provides vectors, cells, and viruses containing the invention's sequences. Also provided are methods for detecting portions of the AAV Rep inhibito...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/22
CPCC07K14/005C12N15/86C12N2710/10343C12N2750/14122C12N2750/14143C12N2840/102C12N2840/60C12Q1/6897A61K2039/5256C12N9/22A61K38/465C12N9/16C12Y301/00A61P31/12C12N7/00C12N2710/10021C12N2710/10033C12N2710/10041C12N2710/10052
Inventor BAHOU, WADIE F.HEARING, PATRICKSITARAMAN, VARSHA
Owner THE RES FOUND OF STATE UNIV OF NEW YORK
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