Tissue preferential codon modified expression cassettes, vectors containing same, and uses thereof

Patent metadata

Field	Value
Publication number	US-9719106-B2
Application number	US-201414787622-A
Country	US
Kind code	B2
Filing date	Apr 29, 2014
Priority date	Apr 29, 2013
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

Described herein are synonymously altered gene sequences which express protein in differing levels within secretory as compared to non-secretory target tissue. An expression cassette comprising an open reading frame (ORF) for a protein under the control of regulatory sequences which direct expression of the product in cell, which ORF has been modified to preferentially increase expression levels in a selected tissue, wherein the modified ORF is characterized by a triplet frequency of any one of Tables 3-12, 16 or 17.

First claim

The invention claimed is:

1. An expression cassette comprising an open reading frame (ORF) for an antibody construct under the control of regulatory sequences which direct expression of the antibody construct in a muscle cell, which ORF has been modified to preferentially increase expression levels in muscle, wherein the modified ORF has a sequence of ORF 201 (SEQ ID NO: 31).

2. The expression cassette according to claim 1 , wherein the regulatory sequences comprise a tissue specific promoter.

3. A vector comprising the expression cassette according to claim 1 and other genetic elements.

4. The vector according to claim 3 , wherein said vector is a recombinant adeno-associated virus (AAV) having an AAV capsid in which the expression cassette is packaged.

5. The vector according to claim 4 , wherein the AAV capsid is selected from AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh10, AAVrh64R1, AAVrh64R2, and rh8.

6. The vector according to claim 3 , wherein the vector is a plasmid.

7. A recombinant AAV comprising an AAV8 capsid and an expression cassette for an anti-HIV antibody, wherein the expression cassette is adapted for expression in a selected target tissue, said expression cassette comprising a modified ORF having a sequence of ORF 201 (SEQ ID NO: 31).

8. A packaging host cell in culture comprising the expression cassette according to claim 1 .

9. An rAAV production system comprising (a) a packaging host cell culture comprising the expression cassette according to claim 1 , (b) AAV rep sequences, AAV cap sequence, and necessary helper functions.

10. The expression cassette according to claim 1 , wherein the expression cassette comprises AAV inverted terminal repeat (ITR) sequences flanking the modified ORF.

11. The recombinant AAV according to claim 7 , further comprising regulatory elements operably linked to the 201 (SEQ ID NO: 31) ORF, which direct expression of the 201 gene product in a host cell.

12. The recombinant AAV according to claim 7 , further comprising AAV inverted terminal repeat (ITR) sequences flanking the ORF.

13. The recombinant AAV according to claim 12 , wherein the ITRs are from a different AAV than the AAV source for the capsid.

Assignees

• Univ Pennsylvania

Inventors

• Wilson James M
• Tretiakova Anna

Classifications

• C12N2830/008

  cell type or tissue specific enhancer/promoter combination · CPC title

• C12N2800/22

  Vectors comprising a coding region that has been codon optimised for expression in a respective host · CPC title

• C12N15/85 Primary

  for animal cells · CPC title

• C12N2750/14143

  viral genome or elements thereof as genetic vector · CPC title

• A61K48/0058

  Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct · CPC title