Polypurine tract modified retroviral vectors
View Patent ↗An integration-defective retroviral vector transfer cassette lacking a functional polypurine tract (PPT) is provided. Also provided are isolated nucleic acids that include a heterologous nucleotide sequence, one or two retroviral long terminal repeats (LTRs), a packaging signal, a rev responsive element, and a eukaryotic promoter, wherein the nucleic acid lacks a functional PPT; vectors that include the disclosed isolated nucleic acids; recombinant retroviral particles and mRNAs thereof; retroviral vector kits; and methods for producing integration-defective vector particles, achieving gene expression of a nucleotide sequence of interest, and inserting a nucleotide sequence of interest into a host cell genome in a site-specific or non-specific manner.
1. A retroviral vector kit comprising:
(a) an integration-defective retroviral RNA vector transfer cassette comprising:
(i) a 5′ long terminal repeat (5′ LTR) and a 3′ long terminal repeat (3′ LTR);
(ii) a packaging signal;
(iii) a primer binding site (PBS);
(iv) a promoter, optionally a eukaryotic promoter; and
(v) a mutation, a deletion, or other modification of the 3′ polypurine tract (3′ PPT),
wherein the 3′ PPT is absent or otherwise non-functional, wherein the retroviral RNA vector transfer cassette is integration defective, and wherein the integration-defective retroviral RNA vector transfer cassette can be reverse-transcribed; and
(b) a packaging cell line comprising at least one construct encoding one or more proteins required for the retroviral RNA vector transfer cassette to be packaged.
2. The retroviral vector kit of claim 1 , wherein the retroviral RNA transfer cassette further comprises a nucleotide sequence of interest (NOI).
3. The retroviral vector kit of claim 2 , wherein the NOI is selected from the group consisting of one or more marker genes, therapeutic genes, antiviral genes, antitumor genes, cytokine genes, genes encoding antigens, sequences that can associate with the host chromatin, sequences that encode a protein that can associate with the host DNA and the host chromatin and the nucleic acid of the vector, sequences that encode a protein having DNA methylation activity and combination thereof.
4. The retroviral vector kit of claim 3 , wherein the one or more marker genes are selected from the group consisting of β-galactosidase gene, hygromycin gene, blastocidin gene, MGMT gene, neomycin gene, puromycin gene, cytosine deaminase gene, secreted alkaline phosphatase gene, fluorescent protein genes, and combinations thereof.
5. The retroviral vector kit of claim 1 , wherein the retroviral RNA transfer cassette further comprises a post-transcriptional regulatory element.
6. A method of producing integration-defective vector particles, comprising introducing into a packaging cell line an integration-defective retroviral RNA vector transfer cassette comprising:
(i) a 5′ long terminal repeat (5′ LTR) and a 3′ long terminal repeat (3′ LTR);
(ii) a packaging signal;
(iii) a primer binding site (PBS);
(iv) a promoter, optionally a eukaryotic promoter; and
(v) a mutation, a deletion, or other modification of the 3′ polypurine tract (3′ PPT),
wherein the 3′ PPT is absent or otherwise non-functional, wherein the retroviral RNA vector transfer cassette is integration defective, and wherein the integration-defective retroviral RNA vector transfer cassette can be reverse-transcribed, wherein the packaging cell line provides proteins for the retroviral RNA vector transfer cassette to be packaged.
7. The method of claim 6 , wherein the retroviral RNA transfer cassette further comprises a nucleotide sequence of interest (NOI).
8. The method of claim 7 , wherein the NOI is selected from the group consisting of one or more marker genes, therapeutic genes, antiviral genes, antitumor genes, cytokine genes, genes encoding antigens, sequences that can associate with the host chromatin, sequences that encode a protein that can associate with the host DNA and the host chromatin and the nucleic acid of the vector, sequences that encode a protein having DNA methylation activity and combination thereof.
9. The method of claim 8 , wherein the one or more marker genes are selected from the group consisting of β-galactosidase gene, hygromycin gene, blastocidin gene, MGMT gene, neomycin gene, puromycin gene, cytosine deaminase gene, secreted alkaline phosphatase gene, fluorescent protein genes, and combinations thereof.
10. The method of claim 6 , wherein the retroviral RNA transfer cassette further comprises a post-transcriptional regulatory element.