Vectors for transgene expression
The present invention relates to a vector system involving replacement of a Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE) sequence with an unrelated short spacer sequence for efficient expression of nucleotides of interest in a retroviral vector system and methods of delivering and expressing nucleotides of interest to target cells.
1. An engineered or non-naturally occurring lentiviral vector comprising a spacer or stuffer nucleotide sequence (SNS) inserted in place of a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), wherein the SNS is heterologous to the vector and does not include a start codon and wherein the SNS is about three to about fifty nucleotides in length.
2. An engineered or non-naturally occurring lentiviral vector containing and expressing a nucleotide sequence of interest (NOI) and a spacer or stuffer nucleotide sequence (SNS) for stimulation of expression of the NOI, wherein the SNS is heterologous to the vector and does not include a start codon, wherein the vector does not contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and wherein the SNS is about three to about fifty nucleotides in length.
3. The vector of claim 1 wherein the SNS is about three to about ten nucleotides in length.
4. The vector of claim 1 wherein the SNS comprises the sequence of SEQ ID NO: 1.
5. The vector of claim 1 , wherein the vector comprises at least one nucleotide sequence of interest (NOI).
6. The vector of claim 1 , wherein the lentiviral vector is a minimal lentiviral vector.
7. The vector of claim 1 , wherein the lentiviral vector is derived from a viral species selected from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), visna/maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).
8. The vector of claim 1 , wherein a nucleic acid sequence encoding Rev is disrupted such that the nucleic acid sequence encodes a non-functional Rev.
9. The vector of claim 1 , wherein a nucleic acid sequence encoding Tat is disrupted such that the nucleic acid sequence encodes a non-functional Tat.
10. The vector of claim 1 , wherein the vector comprises a central polypurine tract (cPPT) sequence.
11. The vector of claim 1 , wherein the vector comprises a gag-packaging signal comprising an ATG motif.
12. The vector of claim 11 , wherein the ATG motif is an ATTG motif.
13. The vector of claim 1 , wherein the vector is multicistronic.
14. The vector of claim 1 , wherein the vector comprises at least one internal regulatory element.
15. The vector of claim 14 , wherein the internal regulatory element is a promoter or an internal ribosomal entry site (IRES).
16. The vector of claim 1 wherein the SNS is about three to about fourteen nucleotides in length.
17. The vector of claim 1 wherein the SNS is about three to about twelve nucleotides in length.
18. The vector of claim 1 wherein the SNS is about three to about nine nucleotides in length.
19. The vector of claim 1 wherein the SNS is about three to about eight nucleotides in length.
20. The vector of claim 1 wherein the SNS is about three to about seven nucleotides in length.
21. The vector of claim 1 wherein the SNS is about three to about five nucleotides in length.