EP0644946A1

EP0644946A1 - Vector particles resistant to inactivation by human serum

Info

Publication number: EP0644946A1
Application number: EP93913964A
Authority: EP
Inventors: W. French Anderson; James M. Mason
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 1992-06-10
Filing date: 1993-05-14
Publication date: 1995-03-29
Also published as: WO1993025698A1; CA2137361A1; JPH09507741A; EP0644946A4

Abstract

A retroviral vector particle resistant to inactivation by human serum. The vector particles preferably include p15E protein wherein at least a portion of the DNA encoding p15E protein is mutated such that the vector particle is resistant to inactivation by human serum. The vector particles may further include a protein containing a receptor binding region which binds to the receptor of a human target cell, thereby enabling the direct introduction of desired heterologous genes in vivo, whereby the vector particle including the heterologous gene travels directly to a targeted cell or tissue.

Description

VECTOR PARTICLES RESISTANT TO INACTIVATION BY HUMAN SERUM

This invention relates to "injectable" vect particles. More particularly, this invention relates vector particles, such as retroviral vector particle wherein such vector particles are resistant to inactivati by human serum.

Vector particles are useful agents for introduci gene(s) or DNA (RNA) into a cell, such as a eukaryotic cel The gene(s) is controlled by an appropriate promote Examples of vectors which may be employed to generate vect particles include prokaryotic vectors, such as bacteri vectors; eukaryotic vectors, including fungal vectors su as yeast vectors; and viral vectors such as DNA vir vectors, RNA virus vectors, and retroviral vector Retroviruses which have been employed for generating vect particles for introducing genes or DNA (RNA) into a ce include Moloney Murine Leukemia Virus, Spleen Necros Virus, Rous Sarcoma Virus and Harvey Sarcoma Virus. T term "introducing" as used herein encompasses a variety methods of transfering genes or DNA (RNA) into a cell. Su methods include transformation, transduction, transfectio and infection.

Vector particles have been used for introducing D (RNA) into cells for gene therapy purposes. In genera such a procedure involves obtaining cells from a patient a using a vector particle to introduce desired DNA (RNA) i the cells and then providing the patient with the engineer cells for a therapeutic purpose. It would be desirable provide alternative procedures for gene therapy. Such alternative procedure would involve genetically engineeri cells jLn vivo. In such a procedure, a vector particle whic includes the desired DNA (RNA) would be administered to patient for .in vivo delivery to the cells of a patient.

It is therefore an object of the present invention t provide gene therapy by introduction of a vector particle such as, for example, a retroviral vector particle, into patient, wherein the vector particle is resistant t inactivation by human serum.

In accordance with an aspect of the present invention there is provided a vector particle which is resistant t inactivation by human serum. Preferably, the vecto particle is a viral vector particle, and more preferably th viral vector particle is a retroviral vector particle.

The envelope portion of retroviruses include a protei known as pl5E, and Applicants have found that retroviruse are susceptible to inactivation by human serum as a resul of the action of complement protein(s) present in serum o the pl5E protein portion of the retroviruε. Applicants hav further found that such retroviruses can be made resistan to inactivation by human serum by mutating such pl5 protein.

In one embodiment, there is provided a retrovira vector wherein a portion of the DNA encoding pl5E protei (shown in the accompanying sequence listing), has bee mutated to render the vector particle resistant t inactivation by human serum. The terms "mutated" an "mutation" as used herein mean that the DNA encoding pl5 protein has been changed such that at least one but not al of the amino acids of pl5E protein have been changed (suc changes can include point mutations, deletions, and/o insertions) . pl5E protein is a viral protein having 196 amino aci residues. In viruses, sometimes all 196 amino acid residue are present, and other times, amino acid residues 181 to 19 (known as the "r" peptide), are not present, and th resulting protein is the "mature" form of pl5E known a pl2E. Thus, viruses can contain both the pl5E and pl2 proteins. pl5E protein is anchored in the viral membran such that amino acid residues 1 to 134 are present on th outside of the virus. Although the present invention is no to be limited to any of the following reasoning, Applicant believe complement proteins may bind to this region whereb such binding leads to inactivation and/or lysis of th retrovirus. In particular, the pl5E protein includes tw regions, amino acid residues 39 to 61 (sometimes hereinafte referred to as region 1), and amino acid residues 101 to 12 (sometimes hereinafter referred to as region 2), whic Applicants believe have an external location in th three-dimensional structure of the pl5E protein; i.e., suc regions are directly exposed to human serum. Region 2 is highly conserved region in many retroviruses, even thoug the amino acid sequences of this region are not identical i all retroviruses. Such regions are complement bindin regions. Examples of complement proteins which may bind t the complement binding regions are CIS and C1Q, which bin to regions 1 and 2.

In order to inactivate the retrovirus, complement protein bind to both region 1 and region 2. Thus, in a preferre embodiment, at least one portion of DNA encoding complement binding region of pl5E protein has been mutated Such a mutation results in a change of at least one amin acid residue of a complement binding region of pl5E protein The change in at least one amino acid residue of complement binding region of pl5E protein prevents bindin of a complement protein to the complement binding region thereby preventing complement inactivation of th retrovirus. In one embodiment, at least one amino aci residue in both complement binding regions of pl5E prote is changed, whereas in another embodiment, at least o amino acid residue in one of the complement binding regio is changed.

It is to be understood, however, that the entire D sequence encoding pl5E protein cannot be mutated becau such a change renders the vectors unsuitable for in vi use.

In one embodiment, the at least one portion of D encoding pl5E protein is mutated such that at least o positively charged amino acid residue or negatively charg amino acid residue is changed to an amino acid resid having the opposite charge.

The positively charged amino acids are His, Lys, a Arg.

The negatively charged amino acids are Asp and Glu.

In another embodiment, the at least one portion of D encoding pl5E protein is mutated such that at least o positively charged amino acid or negatively charged ami acid is changed to a noncharged amino acid.

In one embodiment, the at least one portion of D encoding a complement binding region of pl5E protein, whi is mutated, encodes one or more of amino acid residues 1 to 123 of pl5E protein. In one embodiment, the at least o portion of DNA encoding pl5E protein is mutated such th amino acid residue 122 is changed.

In one embodiment, the at least one portion of D encoding pl5E protein is mutated such that at least one amino acid residues 117 and 122 are changed. Preferabl amino acid residue 117 is changed from Arg to Glu, and ami acid residue 122 is changed from Glu to Gin. In another embodiment, the at least one portion of D encoding pl5E protein^~Yts mutated such that amino ac residues 104, 105, 109, and 111 are changed. Preferabl amino acid residue 104 is changed from Arg to His, ami acid residue 105 is changed from Asp to Asn, amino ac residue 109 is changed from Lys to Gin, and amino ac residue ill is changed from Arg to Gin.

In another embodiment, the at least one portion of D encoding pl5E protein is mutated such that amino ac residues 104, 105, 109, 111, 117, and 122 are change Preferably, the at least one portion of DNA is mutated su that amino acid residue 104 is changed from Arg to Hi amino acid residue 105 is changed from Asp to Asn, ami acid residue 109 is changed from Lys to Gin, amino ac residue 111 is changed from Arg to Gin, amino acid resid 117 is changed from Arg to Glu, and amino acid residue 1 is changed from Glu to Gin.

In yet another alternative embodiment, the mutation DNA encoding pl5E protein may be effected by deleting portion of the pl5E gene, and replacing the deleted porti of the pl5E gene, with fragment(s) or portion(s) of a ge encoding another viral protein. In one embodiment, o portion of DNA encoding the pl5E protein is replaced with fragment of the gene encoding the p21 protein, which is HTLV-I transmembrane protein. HTLV-I virus has been fou to be resistant to binding by complement proteins and th HTLV-I is resistant to inactivation by human serum (Hoshin et al., Nature, Vol. 310, pgs. 324-325 (1984)). Thus, one embodiment, there is also provided a retroviral vect particle wherein a portion of the pl5E protein has be deleted and replaced with a portion of another vir protein, such as a portion of the p21 protein. p21 protein (as shown in the accompanying sequenc listing) is a protein having 176 amino acid residues, an which, in relation to pl5E, has significant amino aci sequence homology. In one embodiment, at least amino aci residues 39 to 61, and 101 to 123 are deleted from pl5 protein, and replaced with amino acid residues 34 to 56 an 96 to 118 of p21 protein. In one alternative, at leas amino acid residues 39 to 123 of pl5E protein are delete and replaced with amino acid residues 34 to 118 of p2 protein.

In another embodiment, amino acid residues 39 to 69 o pl5E protein are deleted and replaced with amino aci residues 34 to 64 of p21 protein, and amino acid residues 9 to 123 of pl5E protein are deleted and replaced with amin acid residues 91 to 118 of p21 protein.

Vector particles thus generated, and which ar resistant to inactivation by human serum, may be engineere such that the vector particles may, when introduced into patient, travel directly to a target cell or tissue. Thus in a preferred embodiment, the vector particle furthe includes a protein which contains a receptor binding regio that binds to a receptor of a human target cell, such as for example, but not limited to, the amphotropic cel receptor.

The retroviral vectors hereinabove described, may b constructed by genetic engineering techniques known to thos skilled in the art.

In one embodiment, the retroviral vector may be of th LN series of vectors, as described in Bender, et al. J.Virol., Vol. 61, pgs. 1639-1649 (1987) and Miller, et al. Biotechniques, Vol. 7, pgs. 980-990 (1989).

In another embodiment, the retroviral vector includes multiple restriction enzyme site, or multiple cloning site The multiple cloning site includes at least four cloning, o restriction enzyme sites, wherein at least two of the site have an average frequency of appearance in eukaryotic gene of less than once in 10,000 base pairs; i.e., th restriction product has an average size of at least 10,00 base pairs.

In general, such restriction sites, also sometime hereinafter referred to as "rare" sites, which have a average frequency of appearance in eukaryotic genes of les than once in 10,000 base pairs, contain a CG doublet withi their recognition sequence, such doublet appearin particularly infrequently in the mammalian genome. Anothe measure of rarity or scarcity of a restriction enzyme sit in mammals is its representation in mammalian viruses, suc as SV40. In general, an enzyme whose recognition sequenc is absent in SV40 may be a candidate for being a "rare mammalian cutter.

Examples of restriction enzyme sites having an averag frequency of appearance in eukaryotic genes of less tha once in 10,000 base pairs include, but are not limited t the NotI, SnaBI, Sail, Xhol, Clal, Sad, EagI, and Sma sites. Preferred cloning sites are selected from the grou consisting of NotI, SnaBI, Sail, and Xhol.

Preferably, the multiple cloning site has a length n greater than about 70 base pairs, and preferably no greate than about 60 base pairs. In general, the multipl restriction enzyme site, or multiple cloning site is locate between the 5'LTR and 3' TR of the retroviral vector. Th 5' end of the multiple cloning site is no greater than abou 895 base pairs from the 3' end of the 5' LTR, and preferabl at least about 375 base pairs from the 3' end of the 5' LTR The 3* end of the multiple cloning site is no greater tha about 40 base pairs fro... the 5¹ end of the 3' LTR, an preferably at least 11 base pairs from the 5' end of the 3 LTR.

Such vectors may be engineered from existing retrovira vectors through genetic engineering techniques known in th art such that the resulting retroviral vector includes a least four cloning sites wherein at least two of the clonin sites are selected from the group consisting of the NotI SnaBI, Sail, and Xhol cloning sites. In a preferre embodiment, the retroviral vector includes each of the NotI SnaBI, Sail, and Xhol cloning sites.

Such a retroviral vector may serve as part of a clonin system for the transfer of genes to eukaryotic cells. Thus there may be provided a cloning system for the manipulatio of genes in a retroviral vector which includes a retrovira vector including a multiple cloning site of the typ hereinabove described, and a shuttle cloning vector whic includes at least two cloning sites which are compatibl with at least two cloning sites selected from the grou consisting of NotI, SnaBI, Sail, and Xhol located on th retroviral vector. The shuttle cloning vector also include at least one desired gene which is capable of bein transferred from said shuttle cloning vector to sai retroviral vector.

The shuttle cloning vector may be constructed from basic "backbone" vector or fragment to which are ligated on or more linkers which include cloning or restriction enzym recognition sites. Included in the cloning sites are th compatible, or complementary cloning sites hereinabov described. Genes and/or promoters having ends correspondin to the restriction sites of the shuttle vector may b ligated into the shuttle vector through techniques known i the art. The shuttle cloning vector can be employed to amplif DNA sequences in prokaryotic systems. The shuttle cloni vector may be prepared from plasmids generally used i prokaryotic systems and in particular in bacteria. Thus for example, the shuttle cloning vector may be derived fro plasmids such as pBR322; pUClθ; etc.

It is also contemplated that within the scope of t present invention that the DNA encoding pl5E protein whic has been mutated to render a vector particle resistant t inactivation by human serum, may be contained in a expression vehicle other than a retroviral vector. Suc expression vehicles include, for example, viral vecto other than retroviral vectors, or any expression plasmi which is capable of being transferred into a cell line whic is capable of producing vector particles which include t mutated pl5E protein.

Such vectors or expression vehicles which contain D encoding a mutated env protein such as the mutated pl5 protein hereinabove described, are transferred into pre-packaging cell line to generate vector particles. I general, the pre-packaging cell line contains the gag an pol proteins of the virus, plus a retroviral vector lackin the structural gag, pol, and env proteins. An example o such a pre-packaging cell line is the GPL pre-packaging cel line which consists of an NIH 3T3 mouse fibroblast cell li which contains an expression plasmid for MoMuLV gag-po protein as well as the retroviral vector LNL6 (Miller, al., Biotechniques, Vol. 7, pgs. 980-990 (1989)). It is be understood, however, that the scope of the prese invention is not to be limited to any particul pre-packaging cell line.

Upon transfection of the pre-packaging cell line wi an expression vehicle containing DNA encoding a mutated e protein, the pre-packaging cell line will generate vector particles. The vector particles are then tested for complement resistance. The vector particles which are shown to be complement resistant (i.e., not inactivated by human serum), therefore, contain complement resistant envelope proteins encoded by a specific envelope expression vehicle. Such an expression vehicle can then be used, by techniques known to those skilled in the art, to produce a packaging cell line which contains an expression vehicle encoding the retroviral gag and pol proteins, and an expression vehicle containing a gene encoding the mutated env protein (such as, for example, an expression vehicle or expression plasmid containing a mutated pl5E protein such as hereinabove described), whereby such packaging cell line may be employed to generate vector particles which are resistant to inactivation by human serum. In particular, a retroviral vector which lacks the structural gag, pol, and env genes, but includes a desired gene of interest, may be transferred into such a packaging cell line. Thus, the packaging cell line may generate vector particles which contain a desired gene(s) of interest, and which are resistant to inactivation by human serum.

The vector particles generated from the packaging cell line will not be inactivated when contacted with human serum; and in addition, such vector particles, when engineered with protein containing a receptor binding region for a human receptor, are targetable, whereby the receptor binding region for a human receptor enables the vector particles to bind to a target cell. Thus, such retroviral vector particles may be directly introduced into the body (e.g., by intravenous, intramuscular, or subcutaneous injection, intranasally, orally, rectally or vaginally), and travel to a desired target cell. Such vector particles, therefore, are useful for the introduction of desire heterologous genes into target cells in vivo as a gen therapy procedure.

Thus, preferably, the vectors of the present inventio further include at least one heterologous gene Heterologous or foreign genes which may be placed into th vector or vector particles include, but are not limited to genes which encode cytokines or cellular growth factors such as lymphokines, which are growth factors fo lymphocytes. Other examples of foreign genes include, bu are not limited to, genes encoding Factor VIII, Factor IX tumor necrosis factors (TNF's), ADA, ApoE, ApoC, and Protei C.

The vectors of the present invention include one o more promoters. Suitable promoters which may be employe include, but are not limited to, the retroviral LTR; th SV40 promoter; and the human cytomegalovirus (CMV) promote described in Miller, et al., Biotechniques, Vol. 7, No. 9 pgs. 980-990 (1989), or any other promoter (eg., cellula promoters such as eukaryotic cellular promoters including but not limited to, the histone, pol III, and β-acti promoters). Other viral promoters which may be employe include, but are not limited to adenovirus promoters, T promoters, and B19 parvovirus promoters. The selection of suitable promoter will be apparent to those skilled in th art from the teachings contained herein.

The vectors of the present invention may contai regulatory elements, where necessary, to ensure tissu specific expression of the desired heterologous gene(s) and/or to regulate expression of the heterologous gene(s) i response to cellular or metabolic signals.

Although the invention has been described with respec to retroviral vector particles, other viral vector particle (such as, for example, adenovirus and adeno-associated vir particles), or synthetic particles may be construct wherein a region of the envelope protein in the vect particle may be mutated such that the vector partic becomes resistant to inactivation by human serum, there making such vector particles suitable for in vi administration.

The invention will now be described with respect to t following example; however, the scope of the prese invention is not intended to be limited thereby.

Example

Plasmid pCE2 was constructed from pBR322 such that t resulting plasmid pCE2 includes genes encoding the envelo proteins gp70 and pl5E. pBR322 (Figure 1) was cut wi EcoRI and filled in to destroy the EcoRI site to gi pBR322Z Rl. pBR3224Rl was then cut with Ndel and filled to destroy the Ndel site to give pBR322 A R pBR322_4 R N was digested with Hindi11 and EcoRV, a cloned into the Hindlll/EcoRV fragment was a Hindlll/Fs cassette containing the gp70 and pl5E genes under t control of a cytomegalovirus (CMV) intermediate ear promoter with a polyA (adenine) tail from SV40 (Figure from plasmid pCEE. (Figure 3). The Hindlll/Fspl casset obtained from plasmid pCEE contains a CMV intermediate ear promoter in which the Ball/Sac11 (bp 21 to bp 766) w converted to an Hindlll/Sall fragment by linker additio the ecotropic envelope Bglll/Nhel fragment (bp 5408 to 7847 of MoMuLV, encoding gp70 and pl5E) was filled and Eco linkers were added; and the SV40 poly A signal from Bell BamHI (bp 2770 to bp 2533) was cloned into a BamHI si (thereby destroying the Bell site). A Bglll site was add at the 3¹ end of the gp70 gene. (This addition does n alter any amino acids). The resulting plasmid is pCE (Figure 4. )

To create the mutations in the pl5E gene, sub-loning i carried out in a different plasmid called pUC-E2 (Figure 6.) This plasmid is pUC18 (Figure 5) with the PvuI fragment removed and replaced with EcoRI linkers. Such wa accomplished by digesting pUC18 with PvuII to remove a 32 bp PvuII fragment, and EcoRI linkers were then added. Int the EcoRI site was cloned the MoMuLV ecotropic envelope gen (i.e., the gp70 and pl5E genes from pCE2) from the Bgll site (5408) to the Nhel site (7847), which have been blunte and had EcoRI linkers added. The resulting pUC-E2 plasmi (Figure 6) therefore has unique Bglll, Spel, Clal, and PvuI sites in and around the pl5E gene.

PCR primers are then synthesized to encode th following mrtations in the pl5E protein (using pCE2 as template):

1. Amino acid residue 117 is changed from Arg to Glu and amino acid residue 122 is changed from Glu to Gin;

2. Amino acid residue 104 is changed from Arg to His amino acid residue 105 is changed from Asp to Asn, amin acid residue 109 is changed from Lys to Gin, and amino aci residue 111 is changed from Arg to Gin; and

3. Amino acid residue 104 is changed from Arg to His amino acid residue 105 is changed from Asp to Asn, amin acid residue 109 is changed from Lys to Gin, amino aci residue 111 is hanged from Arg to Gin, amino acid residu 117 is changed from Arg to Glu, and amino acid residue 12 is changed from Glu to Gin.

Each PCR product is digested with Spel and PvuII, a cloned into pUC-E2 at the unique Spel and PvuII sites. T resulting plasmids are then sequenced to confirm the poi mutations. DNA fragments bearing these mutations are the subcloned into the expression plasmid pCE2. pCE2 i digested with EcoRI and the envelope DNA fragment is remove and replaced with the EcoRI envelope fragment from th pUC-E2 plasmids. The resulting pCE2 plasmids are the checked for orientation of the EcoRI fragment and sequence again (only at the cloning site junctions and at the region bearing the point mutations) to confirm the presence of th newly created mutated pl5E genes. The resulting expressio plasmids are identified as follows: pCR68 - includes mutations in which amino acid residu 117 is changed from Arg to Glu, and amino acid residu 122 is changed from Glu to Gin; pCR69 - includes mutations in which amino acid residu 104 is changed from Arg to His, amino acid residue 10 is changed from Asp to Asn, amino acid residue 109 i changed from Lys to Gin, and amino acid residue 111 i changed from Arg to Gin; and pCR70 - includes mutations in which amino acid residu 104 is changed from Arg to His, amino acid residue 10 is changed from Asp to Asn, amino acid residue 109 i changed from Lys to Gin, amino acid residue 111 i changed from Arg to Gin, amino acid residue 117 i changed from Arg to Glu, and amino acid residue 122 i changed from Glu to Gin.

Plasmids pCR68, pCR69, and pCR70 are transfecte separately into the GPL pre-packaging cell line. The GP pre-packaging cell line consists of an NIH 3T3 mous fibroblast cell line which contains an expression plasmi for MoMuLV gag-pol protein as well as the retroviral vecto LNL6 (Miller, et al., 1989). Upon transfection with pCR68 pCR69, or pCR70, the GPL packaging cell line produces vecto particles. Transiently expressed vector particles are collecte with cell supernatant at 48-72 hrs. post-transfection.

The vector particles generated as hereinabove describ may then be assayed for vector titer by techniques known those skilled in the art. The vector particles may also collected in viral supernatant and concentrated, i necessary, according to procedures known to those skilled i the art in order to employ such vector particles in assa or in therapeutic procedures.

Advantages of the present invention include the abili to introduce vector particles directly into a human patie whereby the vector particle is not lysed or inactivated human serum upon such introduction. Thus, the vect particles of the present invention enable one to deliv desired genes to a patient in vivo. Such vector particl may also be engineered such that they are "targetable", well as injectable, thereby enabling the vector particles travel directly to a target cell or tissue without bei lysed or inactivated by human serum.

It is to be understood, however, that the scope of t present invention is not to be limited to the specifi embodiments described above. The invention may be practic other than as particularly described and still be within t scope of the accompanying claims.

SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: Anderson, W. French

Mason, James M. (ii) TITLE OF INVENTION: Vector Particles

Resistant to Inactivatio by Human Serum (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Carella, Byrne, Bain,

Gilfillan, Cecchi & Stewart

(B) STREET: 6 Becker Farm Road

(C) CITY: Roseland

(D) STATE: New Jersey

(E) COUNTRY: USA

(F) ZIP: 07068 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 inch diskette

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: PC-DOS

(D) SOFTWARE: DW4.V2 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Olεtein, Elliot M.

(B) REGISTRATION NUMBER: 24,025

(C) REFERENCE/DOCKET NUMBER: 271010-73 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 196 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: pl5E protein, (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Glu Pro Val Ser Leu Thr Leu Ala Leu Leu

5 10

Leu Gly Gly Leu Thr Met Gly Gly lie Ala

15 20

Ala Gly lie Gly Thr Gly Thr Thr Ala Leu

25 30

Met Ala Thr Gin Gin Phe Gin Gin Leu Gin

35 40

Ala Ala Val Gin Asp Asp Leu Arg Glu Val

45 50

Glu Lys Ser lie Ser Asn Leu Glu Lys Ser

55 60

Leu Thr Ser Leu Ser Glu Val Val Leu Gin

65 70

Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu

75 80

Lys Glu Gly Gly Leu Cyε Ala Ala Leu Lys

85 90

Glu Glu Cys Cys Phe Tyr Ala Asp His Thr

95 100 Gly Leu Val Arg Asp Ser Met Ala Lys Leu

105 110

Arg Glu Arg Leu Asn Gin Arg Gin Lys Leu

115 120

Phe Glu Ser Thr Gin Gly Trp Phe Glu Gly

125 130

Leu Phe Asn Arg Ser Pro Trp Phe Thr Thr

135 140

Leu lie Ser Thr lie Met Gly Pro Leu lie

145 150

Val Leu Leu Met lie Leu Leu Phe Gly Pro

155 160

Cys lie Leu Asn Arg Leu Val Gin Phe Val

165 170

Lys Asp Arg lie Ser Val Val Gin Ala Leu

175 180

Val Leu Thr Gin Gin Tyr His Gin Leu Lys

185 190

Pro lie Glu Tyr Glu Pro

195

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 176 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE

(A) NAME/KEY: p21 protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ala Val Pro Val Ala Val Trp Leu Val Ser

5 10

Ala Leu Ala Met Gly Ala Gly Val Ala Gly

15 20

Arg lie Thr Gly Ser Met Ser Leu Ala Ser

25 30

Gly Lys Ser Leu Leu His Glu Val Asp Lys

35 40

Asp lie Ser Gin Leu Thr Gin Ala lie Val

45 50

Lys Asn His Lys Asn Leu Leu Lys lie Ala

55 60

Gin Tyr Ala Ala Gin Asn Arg Arg Gly Leu

65 70

Asp Leu Leu Phe Trp Glu Gin Gly Gly Leu

75 80

Cyε Lys Ala Leu Gin Glu Gin Cys Cys Phe

85 90

Leu Asn lie Thr Asn Ser His Val Ser lie

95 100

Leu Gin Glu Arg Pro Pro Leu Glu Asn Arg

105 110

Val Leu Thr Gly Trp Gly Leu Asn Trp Asp

115 120

Leu Gly Leu Ser Gin Trp Ala Arg Glu Ala

125 130

Leu Gin Thr Gly lie Thr Leu Val Ala Leu

135 140

Leu Leu Leu Val lie Leu Ala Gly Pro Cys

145 150 lie Leu Arg Gin Leu Arg His Leu Pro Ser

155 160 Arg Val Arg Tyr Pro Hiε Tyr -Ser Leu lie

165 170

Asn Pro Glu Ser Ser Leu

175

Claims

WHAT IS CLAIMED IS:

1. A retroviral vector particle, said vect particle being resistant to inactivation by human serum.

2. The vector particle of Claim 1 wherein sa vector particle includes pl5E protein, and wherein a porti but not all of the pl5E protein has been mutated to rend the vector particles reεistant to inactivation by hum serum.

3. The vector particle of Claim 2 wherein portion of the pl5E protein has been mutated such that least one amino acid of pl5E protein has been changed.

4. The vector particle of Claim 3 wherein at lea one portion of a complement binding region of pl5E prote has been mutated.

5. The vector particle of Claim 4 wherein sa complement binding region is selected from the gro consisting of amino acid residues 39 to 61 and 101 to 123 pl5E protein.

6. The vector particle of Claim 5 wherein sa complement binding region is amino acid residues 101 to 1 of pl5E protein.

7. The vector particle of Claim 6 wherein pl protein is mutated such that amino acid residue 122 of pl protein is changed.

8. The vector particle of Claim 6 wherein pl protein is mutated such that amino acid residues 117 and 1 are changed.

9. The vector particle of Claim 8 wherein pl protein is mutated such that amino acid residue 117 changed from Arg to Glu, and amino acid residue 122 changed from Glu to Gin.

10. The vector particle of Claim 6 wherein pl5 protein is mutated such that amino acid residues 104, 105 109, and 111 are changed.

11. The vector particle of Claim 10 wherein pl5 protein is mutated such that amino acid residue 104 i changed from Arg to His, amino acid residue 105 is chang from Asp to Asn, amino acid residue 109 is changed from L to Gin, and amino acid residue 111 is changed from Arg t Gin.

12. The vector particle of Claim 6 wherein pl5 protein is mutated such that amino acid residues 104, 105 109, 111, 117, and 122 are changed.

13. The vector particle of Claim 12 wherein pl protein is mutated such that amino acid residue 104 i changed from Arg to His, amino acid residue 105 is chang from Asp to Asn, amino acid residue 109 is changed from L to Gin, amino acid residue 111 is changed from Arg to Gi amino acid residue 117 is changed from Arg to Glu, and ami acid residue 122 is changed from Glu to Gin.

14. The vector particle of Claim 5 wherein pl5 protein is mutated such that at least one positively o negatively charged amino acid is changed to an amino aci having the opposite charge.

15. The vector particle of Claim 5 wherein pl5 protein is mutated such that at least one positively charg or negatively charged amino acid is changed to a non-charg amino acid.

16. The vector particle of Claim 1 wherein sai vector particle further includes a protein containing receptor binding region that binds to a receptor of a hum target cell.

17. The vector particle of Claim 16 and furthe containing a heterologous gene.

18. Eukaryotic cells transformed with the vecto particles of Claim 17.

19. An expression vehicle including DNA encodin pl5E protein, wherein said DNA encoding pl5E protein i mutated such that when said mutated pl5E protein encoded b said DNA is included in a vector particle, said vecto particle is resistant to inactivation by human serum.

20. A packaging cell line including the expreεsio vehicle of Claim 19.

21. A viral vector particle, said viral vecto particle being resistant to inactivation by human serum.