Vectors containing HIV packaging sequences, packaging defective HIV vectors, and uses thereof

Claim 2

An HIV vector comprising: (a) a DNA segment from an HIV genome, wherein the DNA segment comprises the HIV gag, pol and env genes; wherein said HIV vector lacks the HIV packaging sequence necessary to package HIV RNA into virions; wherein said HIV packaging sequence is the nucleotide sequence located between the 5' major splice donor site and the initiation codon of the gag gene on the HIV genome; and (b) a promoter operably linked to the DNA segment from an HIV genome of (a); wherein the HIV vector, when introduced into a eukaryotic host cell, express HIV gag, pol and env proteins to form HIV virions that do not contain sufficient HIV RNA to result in a replication competent HIV virion.

Claim 3

The HIV vector claim 1, wherein the promoter is an HIV LTR.

Claim 4

The HIV vector of claim 1, wherein the promoter functions to express genes preferentially in specific cell types.

Claim 5

The HIV vector claim 3, wherein the promoter is a CMV promoter.

Claim 6

The HIV vector of claim 1, wherein the HIV packaging sequence is a nucleotide sequence located from the 5' major splice donor site to about 5 bases upstream of the gag gene initiation codon.

Claim 7

The HIV vector of claim 1, wherein the HIV packaging sequence is a nucleotide sequence located from about 9 bases downstream of the 5' major splice donor site to about 14 bases upstream of the gag gene initiation codon.

Claim 8

The HIV vector of claim 1, wherein the HIV packaging sequence is the sequence AAAAATTTTGACTAGCGGA.

Claim 9

The HIV vector of claim 1, wherein the HIV genome is an HIV-1 genome.

Claim 10

Thee HIV vector of claim 7, wherein the HIV genome is an HIV-1 genome.

Claim 11

The HIV vector of claim 8, wherein the promoter is an HIV-1 LTR.

Claim 12

The HIV vector of claim 9, wherein the promoter is an HIV-1 LTR.

Claim 13

At least two HIV vectors, wherein each of the at least two HIV vectors has (a) a DNA segment encoding a gag or an env gene from an HIV genome but not both, wherein one of the at least two HIV vector or an additional HIV vector has a DNA segment encoding a pol gene from an HIV genome, wherein each of the HIV vectors lacks the HIV packaging sequence necessary to package HIV RNA into virions; wherein said HIV packaging sequence is the nucleotide sequence located between the 5' major splice donor site and the initiation codon of the gag gene on the HIV genome; (b) a promoter operably linked to each of the DNA segments from an HIV genome of (a); and (c) a polyadenylation sequence located downstream of each of the DNA segments from an HIV genome of (a); wherein the polyadenylation sequence does not comprise a functional LTR sequence; wherein the vectors, when introduced into a eukaryotic host cell, express in combination the gag, pol and env proteins to form HIV virions that do not contain sufficient HIV RNA to result in a replication competent HIV virion.

Claim 14

At least two HIV vectors, wherein each of the at least two HIV vectors has (a) a DNA segment encoding a gag or an env gene from an HIV genome but not both, wherein one of the at least two HIV vector or an additional HIV vector has a DNA segment encoding a pol gene from an HIV genome, wherein each of the HIV vectors lacks the HIV packaging sequence necessary to package HIV RNA into virions; wherein said HIV packaging sequence is the nucleotide sequence located between the 5' major splice donor site and the initiation codon of the gag gene on the HIV genome; (b) a promoter operably linked to each of the DNA segments from an HIV genome of (a); and (c) a polyadenylation sequence located downstream of each of the DNA segments from an HIV genome of (a); wherein the polyadenylation sequence does not comprise a functional LTR sequence; wherein the vectors, when introduced into a eukaryotic host cell, express in combination the gag, pol and env proteins to form HIV virions that do not contain sufficient HIV RNA to result in a replication competent HIV virion.

Claim 15

Two HIV vectors, wherein each vector has inserted therein: (a) exactly one of two of any of three DNA segments from an HIV genome, wherein the three DNA segments are selected from the group consisting of the HIV gag, pol and env genes; wherein each of the two vectors lacks the HIV packaging sequence necessary to package HIV RNA into virions; wherein said HIV packaging sequence is the nucleotide sequence located between the 5' major splice donor site and the initiation codon of the gag gene on the HIV genome; (b) a promoter operably linked to each of the DNA segments from an HIV genome of (a); and (c) a polyadenylation sequence located downstream of each of the DNA segments from an HIV genome of (a); wherein the polyadenylation sequence does not comprise a functional LTR sequence; wherein the two vectors, when introduced into a eukaryotic host cell, express in combination the gag, pol and env proteins to form HIV virions that do not contain sufficient HIV RNA to result in a replication competent HIV virion.

Claim 16

Two HIV vectors, wherein each vector has inserted therein: (a) exactly one of two of any of three DNA segments from an HIV genome, wherein the three DNA segments are selected from the group consisting of the HIV gag, pol and env genes; wherein each of the two vectors lacks the HIV packaging sequence necessary to package HIV RNA into virions; wherein said HIV packaging sequence is the nucleotide sequence located between the 5' major splice donor site and the initiation codon of the gag gene on the HIV genome; (b) a promoter operably linked to each of the DNA segments from an HIV genome of (a); and (c) a polyadenylation sequence located downstream of each of the DNA segments from an HIV genome of (a); wherein the polyadenylation sequence does not comprise a functional LTR sequence; wherein the two vectors, when introduced into a eukaryotic host cell, express in combination the gag, pol and env proteins to form HIV virions that do not contain sufficient HIV RNA to result in a replication competent HIV virion.

Claim 17

The two HIV vectors of claim 13, wherein either or both vectors further comprise at least one HIV regulatory gene which expresses a functional HIV regulatory protein in said eukaryotic host cell.

Claim 18

The two HIV vectors of claim 14, wherein said HIV regulatory gone is selected from the group consisting of tat, rev, vpu, vpr and vif.

Claim 19

The two HIV vectors of claim 13, wherein the HIV vector containing the HIV env gene does not also contain the gag gene.

Claim 20

The two HIV vectors of claim 13, wherein the promoter of (b) is an HIV LTR.

Claim 21

The two HIV vectors of claim 13, wherein the HIV packaging sequence is a nucleotide sequence located from the 5' major splice donor site to about 5 bases upstream of the gag initiation codon.

Claim 22

The two HIV vectors of claim 13, wherein the HIV genome is selected from the group consisting of HIV-1 and HIV-2.

Claim 23

The two HIV vectors of claim 13, wherein the HIV genome is the HIV-1 genome.

Claim 24

The two HIV vectors of claim 13, wherein the HIV genome is the HIV-2 genome.

Claim 25

The two HIV vectors of claim 13, wherein the first of the two HIV vectors contains both the HIV gag and the HIV pol genes.

Claim 26

The two HIV vectors of claim 22, wherein the second of the two HIV vectors contains the env gene.

Claim 27

The two HIV vectors of claim 22, wherein the promoter for the first of the two HIV vectors is an HIV LTR.

Claim 28

The two HIV vectors of claim 23, wherein the promoter for the second of the two HIV vectors is an HIV LTR.

Claim 29

The two HIV vectors of claim 23, wherein the polyadenylation sequence is the SV40 polyadenylation sequence.

Claim 30

The two HIV vectors of claim 22, wherein the polyadenylation sequence for the first of the two HIV vectors is the SV40 polyadenylation sequence.

Claim 31

The two HIV vectors of claim 23, wherein the polyadenylation sequence for the second of the two HIV vectors is the SV40 polyadenylation sequence.

Claim 32

The two HIV vectors of claim 28, wherein the second of the two HIV vectors further comprises an HIV rev gene which expresses a functional HIV rev protein in said eukaryotic host cell.

Claim 33

The two HIV vectors of claim 13 wherein each of the two HIV vectors further comprises an HIV RRE sequence.

Claim 34

Three vectors, wherein the first two vectors consist of the two HIV vectors of claim 13, and the third vector is a retroviral vector which comprises: (i) a gene of interest, wherein said gene is heterologous to an HIV genome; (ii) a promoter operably linked to the gene of (i); (iii) an HIV packaging sequence necessary to package HIV RNA into HIV virions; and (iv) single HIV LTRs flanking the sequences of (i), (ii) and (iii), wherein the HIV LTRs are the 5' HIV LTR and the 3' HIV LTR which permit reverse transcription and integration of the third vector in a eukaryotic host cell; wherein the first two HIV vectors, when introduced into a eukaryotic host cell, express in combination the HIV gag, pol and env proteins such that when the third vector is also introduced into said host cell, the HIV gag, pol and env proteins will package the third vector into infectious HIV vector virions.

Claim 35

The three vectors of claim 31, wherein the gene of (i) further comprises a polyadenylation sequence located downstream of said gene.

Claim 36

The three vectors of claim 31, wherein the third vector further comprises a second gene heterologous to an HIV genome.

Claim 37

The three vectors of claim 31, wherein the HIV packaging sequence of (iii), present in the third vector, is the nucleotide sequence located between the 5' major splice donor site and up to at most nucleotide position 385 located in the 5' portion of the gag gene on the HIV-1 genome.

Claim 38

The three vectors of claim 31, wherein the HIV packaging sequence of (iii), present in the third vector, is the nucleotide sequence located between the 5' major splice donor site and about nucleotide position 338 to 385 located in the 5' portion of the gag gene on the HIV-1 genome.

Claim 39

The three vectors of claim 31, wherein the HIV packaging sequence of (iii), present in the third vector, is the nucleotide sequence located between the 5' major splice donor site and about nucleotide position 350 to 381 located in the 5' portion of the gag gene on the HIV-1 genome.

Claim 40

The three vectors of claim 31, wherein the HIV packaging sequence of (iii), present in the third vector, is the nucleotide sequence located between the 5' major splice donor site and a Bal I restriction site at the 3' end of the gag gene of the HXBc2 strain of HIV-1.

Claim 41

The three vectors of claim 31, wherein the promoter of (ii) is a viral promoter.

Claim 42

The three vectors of claim 38, wherein the viral promoter is the SL3 promoter.

Claim 43

The three vectors of claim 31, wherein the gene of (i) encodes a trans-dominant inhibitor protein, an antisense RNA, a catalytic RNA or a soluble CD4 protein.

Claim 44

The three vectors of claim 31, wherein the promoter of (ii) is the 5' HIV LTR of (iv).

Claim 45

The three vectors of claim 31, wherein the promoter of (ii) is a promoter heterologous to an HIV genome.

Claim 46

Two HIV vectors wherein (a) one HIV vector, referred to as said first vector, contains a DNA segment from an HIV genome consisting of the HIV gag gene or the HIV gag-pol genes, wherein said first vector does not contain a DNA segment containing the HIV env gene, wherein said first vector can optionally contain a DNA segment from an HIV genome consisting of the HIV pol gene, (b) the other HIV vector, referred to as said second vector, contains a DNA segment from an HIV genome consisting of the HIV env gene, wherein said second vector does not contain a DNA segment containing the HIV gag gene, wherein said second vector can optionally contain a DNA segment from an HIV genome consisting of the HIV pol gene, (c) wherein each of the HIV vectors lacks the HIV packaging sequence necessary to package HIV RNA into virions; wherein said HIV packaging sequence is the nucleotide sequence located between the 5' major splice donor site and the initiation codon of the gag gene on the HIV genome; (d) a promoter operably linked to each of the DNA segments from an HIV genome of (a) and (b); and (e) a polyadenylation sequence located downstream of each of the DNA segments from an HIV genome of (a) and (b); wherein the polyadenylation sequence does not comprise a functional LTR sequence; wherein the two HIV vectors, when introduced into a eukaryotic host cell, express in combination the gag, pol and env proteins to form HIV virions that do not contain sufficient HIV RNA to result in a replication competent HIV virion.

Claim 47

Two HIV vectors wherein (a) one HIV vector, referred to as said first vector, contains a DNA segment from an HIV genome consisting of the HIV gag gene or the HIV gag-pol genes, wherein said first vector does not contain a DNA segment containing the HIV env gene, wherein said first vector can optionally contain a DNA segment from an HIV genome consisting of the HIV pol gene, (b) the other HIV vector, referred to as said second vector, contains a DNA segment from an HIV genome consisting of the HIV env gene, wherein said second vector does not contain a DNA segment containing the HIV gag gene, wherein said second vector can optionally contain a DNA segment from an HIV genome consisting of the HIV pol gene, (c) wherein each of the HIV vectors lacks the HIV packaging sequence necessary to package HIV RNA into virions; wherein said HIV packaging sequence is the nucleotide sequence located between the 5' major splice donor site and the initiation codon of the gag gene on the HIV genome; (d) a promoter operably linked to each of the DNA segments from an HIV genome of (a) and (b); and (e) a polyadenylation sequence located downstream of each of the DNA segments from an HIV genome of (a) and (b); wherein the polyadenylation sequence does not comprise a functional LTR sequence; wherein the two HIV vectors, when introduced into a eukaryotic host cell, express in combination the gag, pol and env proteins to form HIV virions that do not contain sufficient HIV RNA to result in a replication competent HIV virion.

Claim 48

The two HIV vectors of claim 43, wherein either or both HIV vectors further comprise at least one HIV regulatory gene which expresses a functional HIV regulatory protein in said eukaryotic host cell.

Claim 49

The two HIV vectors of claim 44, wherein said HIV regulatory gene is selected from the group consisting of tat, rev, vpu, vpr and vif.

Claim 50

The two HIV vectors of claim 43 wherein each of the two HIV vectors further comprises an HIV RRE sequence.

Claim 51

Three vectors, wherein the first two vectors consist of the two HIV vectors of claim 43, and the third vector is a retroviral vector which comprises: (i) a gene of interest, wherein said gene is heterologous to an HIV genome; (ii) a promoter operably linked to the gene of (i); (iii) an HIV packaging sequence necessary to package HIV RNA into virions; and (iv) single HIV LTRs flanking the sequences of (i), (ii) and (iii), wherein the HIV LTRs are the 5' HIV LTR and the 3' HIV LTR which permit reverse transcription and integration of the third vector in a eukaryotic host cell; wherein the first two HIV vectors, when introduced into a eukaryotic host cell, express in combination the HIV gag, pol and env proteins such that when the third vector is also introduced into said host cell, the HIV gag, pol and env proteins will package the third vector into infectious HIV vector virions.

Claim 52

The three vectors of claim 47, wherein the gene of (i) further comprises a polyadenylation sequence located downstream of said gene.

Claim 53

The three vectors of claim 47, wherein the third vector further comprises a second gene heterologous to an HIV genome.

Claim 54

The three vectors of claim 47, wherein the promoter of (ii) is a viral promoter.

Claim 55

The three vectors claim 50, wherein the viral promoter is the SL3 promoter.

Claim 56

The three vectors of claim 47, wherein the gene of (i) encodes a trans-dominant inhibitor protein, an antisense RNA, a catalytic RNA or a soluble CD4 protein.

Claim 57

Four vectors, wherein the first three vectors consist of the three vectors of claim 47, and the fourth vector is an HIV vector which contains: (f) a DNA segment from an HIV genome consisting of at least one HIV regulatory gene, wherein the fourth vector lacks the HIV packaging sequences necessary to package HIV RNA into virions; wherein said HIV packaging sequence is the nucleotide sequence located between the 51 major splice donor site and the initiation codon of the gag gene on the HIV genome; (g) a promoter operably linked to the DNA segment from the HIV genome of (f), and (h) a polyadenylation sequence located downstream of the DNA segments from the HIV genome of (g), wherein the polyadenylation sequence does not comprise a functional LTR sequence.

Claim 58

The four vectors of claim 53, wherein the fourth vector contains the HIV vpu gene and does not contain the HIV gag and env genes.

Claim 59

A method of producing an HIV packaging cell line which comprises transforming an established cell line with the vectors of claim 12.

Claim 60

A method of producing an HIV packaging cell line which comprises transforming an established cell line with the vector of claim 1.

Claim 61

A method of producing an HIV packaging cell line which comprises transforming an established cell line with the vectors of claim 13.

Claim 62

A method of producing an HIV packaging cell line which comprises transforming an established cell line with the vectors of claim 43.

Claim 63

The method of claim 55, wherein the established cell line is a mammalian cell line.

Claim 64

The method of claim 56, wherein the established cell line is a mammalian cell line.

Claim 65

The method of claim 57, wherein the established cell line is a mammalian cell line.

Claim 66

The method of claim 58, wherein the established cell line is a mammalian cell line.

Claim 67

An HIV packaging cell line which comprises an established cell line transformed by the vectors of claim 12.

Claim 68

An HIV packaging cell line which comprises an established cell line transformed by the vector of claim 1.

Claim 69

An HIV packaging cell line which comprises an established cell line transformed by the vectors of claim 13.

Claim 70

An HIV packaging cell line which comprises an established cell line transformed by the vectors of claim 43.

Claim 71

A method of transferring a gene of interest to a mammalian cell, comprising: (a) transfecting a eukaryotic host cell with the three vectors of claim 31; (b) culturing the transfected host cell of (a) and collecting the packaged virions produced; (c) administering the virions of (b) to a mammalian cell to allow infection of the mammalian cell and transfer of the gene of interest.

Claim 72

A method of transferring a gene of interest to a mammalian cell, comprising: (a) transfecting a eukaryotic host cell with the three vectors of claim 47; (b) culturing the transfected host cell of (a) and collecting the packaged virions produced; (c) administering the virions of (b) to a mammalian cell to allow infection of the mammalian cell and transfer of the gene of interest.

Claim 73

The method of claim 67, wherein the mammalian cell is located in vitro.

Claim 74

The method of claim 68, wherein the mammalian cell is located in vitro.

Claim 75

The method of claim 67, wherein the mammalian cell is located in vivo.

Claim 76

The method of claim 68, wherein the mammalian cell is located in vivo.