Adeno-associated-virus rep sequences, vectors and viruses

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...

AI Technical Summary

Benefits of technology

[0089]The terms “reduce,”“inhibit,”“diminish,”“suppress,”“decrease,” and grammatical equivalents (including “lower,”“smaller,” etc.) when in reference to the level of any molecule (e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.), cell, virus, and / or phenomenon (e.g., expression, transcription, translation, viral infection, viral productive infection, viral replication, viral replication competence, Rep-mediated nuclease activity, site-specific integration into a genome, helicase activity, disease symptom, binding to a molecule, specificity of binding of two molecules, affinity of binding of two molecules, specificity to disease, sensitivity to disease, affinity of binding, enzyme activity, etc.) in a first sample (or in a first subject) relative to a second sample (or relative to a second subject), mean that the quantity of molecule, cell and / or phenomenon in the first sample (or in the first subject) is lower than in the second sample (or in the second subject) by any amount that is statistically significant using any art-accepted statistical method of analysis. In one embodiment, the quantity of molecule, cell and / or phenomenon in the first sample (or in the first subject) is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and / or at least 90% lower than the quantity of the same molecule, cell and / or phenomenon in the second sample (or in the second subject). In another embodiment, the quantity of molecule, cell, and / or phenomenon in the first sample (or in the first subject) is lower by any numerical percentage from 5% to 100%, such as, but not limited to, from 10% to 100%, from 20% to 100%, from 30% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from 70% to 100%, from 80% to 100%, and from 90% to 100% lower than the quantity of the same molecule, cell and / or phenomenon in the second sample (or in the second subject). In one embodiment, the first subject is exemplified by, but not limited to, a subject that has been manipulated using the invention's compositions and / or methods. In a further embodiment, the second subject is exemplified by, but not limited to, a subject that has not been manipulated using the invention's compositions and / or methods. In an alternative embodiment, the second subject is exemplified by, but not limited to, a subject to that has been manipulated, using the invention's compositions and / or methods, at a different dosage and / or for a different duration and / or via a different route of administration compared to the first subject. In one embodiment, the first and second subjects may be the same individual, such as where the effect of different regimens (e.g., of dosages, duration, route of administration, etc.) of the invention's compositions and / or methods is sought to be determined in one individual. In another embodiment, the first and second subjects may be different individuals, such as when comparing the effect of the invention's compositions and / or methods on one individual participating in a clinical trial and another individual in a hospital.

[0091]The terms “alter” and “modify” when in reference to the level of any molecule and / or phenomenon refer to an increase and / or decrease.

[0092]“Substantially the same” when in reference to the level of any molecule (e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.), cell, virus, and / or phenomenon (e.g., expression, transcription, translation, viral infection, viral productive infection, viral replication, viral replication competence, Rep-mediated nuclease activity, site-specific integration into a genome, helicase activity, disease symptom, binding to a molecule, specificity of binding of two molecules, affinity of binding of two molecules, specificity to disease, sensitivity to disease, affinity of binding, enzyme activity, etc.) in a first sample (or in a first subject) relative to a second sample (or relative to a second subject), mean that the quantity of molecule, cell and / or phenomenon in the first sample (or in the first subject) is not different from the quantity in the second sample (or in the second subject) using any art-accepted statistical method of analysis. In one embodiment, the quantity of molecule, cell and / or phenomenon in the first sample (or in the first subject) is from 90% to 100% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) of the quantity in the second sample (or in the second subject).

[0093]Reference herein to any numerical range expressly includes each numerical value (including fractional numbers and whole numbers) encompassed by that range. To illustrate, and without limitation, reference herein to a range of “at least 50” includes whole numbers of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers 50.1, 50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc. In a further illustration, reference herein to a range of “less than 50” includes whole numbers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., and fractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 49.0, etc. In yet another illustration, reference herein to a range of from “5 to 10” includes each whole number of 5, 6, 7, 8, 9, and 10, and each fractional number such as 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, etc.BRIEF SUMMARY OF THE INVENTION

[0094]The AAV Rep78 protein is required for SSI although it displays an inhibitory effect on virus replication in hybrid viruses (e.g., Ad / AAV viruses). To date, prior art strategies to construct hybrid viruses (e.g., Ad / AAV) by controlling Rep expression have met with limited success. The invention provides the discovery that AAV Rep's cis-acting inhibitory effect on hybrid virus replication and / or replication competence and / or infectivity and / or productive infectivity is due to a role of an inhibitory sequence within the Rep ORF.

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.

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