CA2137361A1 - Vector particles resistant to inactivation by human serum - Google Patents
Vector particles resistant to inactivation by human serumInfo
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Abstract
2137361 9325698 PCTABScor01 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
W093/2~698~ PCT/~S93/04706 ~137361 VE~TOR PARTICLES RESISTANT TO INACTIVATION
BY ~nMAN 5ERUM
' ~
This invention relates to "injectable" vector particles. More particularly, this invention relates to vector particles, such as retroviral vector particles, wherein such vector particles are resistant to i~activation by human serum.
Vector particles are u~eful agents for introducing gene(~) or DNA (RNA) into a cell, such as a eukaryotic cell.
The gene(~) is controlled by an appropriate promoter.
Examples of vectors which may be employed to generate vector particles includ~ prokaryotic vectors, such as bacterial vectors; eukaryotic vectors; including fungal vectors such as yeast vectors; and viral vectors such as DNA virus vectors, RNA virus vectors, and retroviral vectors.
Retroviruses which have been employed for generating vector particles for introducing genes or DNA (RNA) into a cell include Moloney Murine Leukemia Virus, Spleen Necrosis Virus, Rous Sarcoma Virus and Harvey Sarcoma Virus. The term "introducing" as u~ed herein encompasses a variety of methods of transfering genes or DNA tRNA) into a cell. Such methods include transformation, transduction, transfection, and infection.
Vector particles have been used for introducing DNA
(RNA) into cells for gene therapy purposes. In general, such a procedure involves obtaining cells from a patient and using a vector particle to introduce desired DNA (RNA) into the cells and then providing the patient with the engineered cells for a therapeutic purpose. It would be desirable to provide alternative procedures for gene therapy. Such an alternative procedure would involve genetically engineering SUBSTITUTE SH~ET (RVLE 26) :
W093/2569~ 2 1 3 7 3 ~ 1 PC~/US93/047~6 r~
cells in vivo. In such a procedure, a vector particle which includes the desired DNA (RNA) would be administered to a patient for in v vo delivery to the cells of a patient.
It is therefore an object of the present invention to provide gene therapy by introduction of a vector particle, uch as, for example, a retroviral vector particle, into a patient, wherein the vector particle is resistant to inactivation ~y human serum.
In accordance with an aspect of the present invention, there is provided a vector particle which is resistant to inactivation by human serum. Preferably, the vector particle is a viral vector particle, and more preferably the viral vector particle is a retroviral vector particle.
The envelope portion of retroviruses include a protein known as pl5E, and Applicants have found that retroviruses are susceptible to inactivation by human serum as a result of the action of complement protein(s) present in serum on the pl5E protein portion of the retrovirus. Applicants have further found that such retroviruses can be made resistant to inactivation by human serum by mutating such pl5E
protein.
In one embodiment, there is provided a retroviral vector wherein a portion of the DNA encoding pl5E protein (shown in the accompanying sequence listing), has been mutated to render the vector particle resistant to inactivation by human serum. The terms "mutated" and "mutation" as used herein mean that the DNA encoding pl5E
protein has been changed such that at least one but not àll of the amino acids of pl5E protein have been changed (such changes can include point mutations, deletions, and/or insertions).
pl5E protein is a viral protein having 196 amino acid residues. ln viruses, sometimes all 196 amino acid residues SUBSlITUTE SHEET (RULE 26) W093/~5698 ~ ~ 3 7 ~ 6 1 PCT/US93/047~6 f~
are present, and other times, amino acid residues 181 to 196 (known as the "r" peptide), are not present, and the resulting protein is the "mature" form of pl5E known as pl2E. Thus, viruses can contain both the pl5E and pl2E
proteins. pl5E protein is anchored in the viral membrane such that amino acid residues 1 to 134 are present on the outside of the virus. Although the present invention is not to be limited to any of the following reasoning, Applicants believe complement proteins may bind to this region whereby such binding leads to inactivation and/or lysis of the retrovirus. In particular, the pl5E protein includes two regions, amino acid residues 39 to 61 (sometimes hereinafter referred to as region 1), and amino acid residues 101 to 123 (sometimes hereinafter referred to as region 2), which Applicants believe have 'an external location in the three-dimensional structure of the pl5E protein; i.e., such regions are directly exposed to human serum. Region 2 is a highly conserved region in many retroviruses, even though the amino acid ~eguences of this region are not identical in all retroviruses. Such regions are complement binding regions. Examples of complement proteins which may bind to the complement binding regions are ClS and ClQ, which bind to regions 1 and 2.
In order to inactivate the retrovirus, complement proteins bind to both region 1 and region 2. Thus, in a preferred embodiment, at least one portion of DNA encoding a complement binding region of pl5E protein has been mutated.
Such a m~ltation results in a change of at least one amino acid residue of a complement binding region of pl5E protein.
The change in at least one amino acid residue of a complement binding region of pl5E protein prevents binding of a complement protein to the complement binding region, thereby preventing complement inactivation of the SUBST~TIJTE SHEET (RULE 26~
W093/~5698 21373~1 PCT/US93/04706 retrovirus. In one embodiment, at least one amino acid residue in both complement binding region~ of pl5E protein is changed, whereas in another embodiment, at least one amino acid residue in one of the complement binding regions i 9 cha~ged.
It is to be understood, howaver, that the entire DNA
se-guence encoding pl5E protein cannot be mutated because such a change renders the vectors unsuitable for in vivo use.
In one embodiment, the at least one portion of DNA
encoding pl5E protein is mutated such that at least one positively charged amino acid residue or negatively charged amino acid residue is changed to an amino acild residue having the opposite charge.
The positively charged amino acids are His, Lys, and Arg.
The negatively charged amino acids are Asp and Glu.
In another~embodiment, the at least one portion of DNA
encoding pl~E protein is mutated such that at least one positively charged amino acid or negatively charged amino acid is changed to a noncharged amino acid.
In one embodiment, the at least one portion of DNA
encoding a complement binding region of pl5~ protein, which is mutatad, encodes one or more of amino acid residues 101 to 123 of pl5E protein. In one em~odiment, the at least one portion of DNA encoding pl5E protein is mutated such that amino acid residue 122 is changed.
In one embodiment, the at least one portion of DNA
encoding pl5E protein is mutated such that at least one of amino acid residues 117~ and 122 are changed. Preferably, amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 i8 changed from Glu to Gln.
SUBSTITUTE SHEET (RULE 26) W093/25698 ~ PCT/US93/04706 In another embodiment, the at least one portion of DNA
encoding pl5~ protein is mutated ~uch that amino acid residues 104, 105, 109, and 111 are changed. Preferably, amino acid residue 104 is changed from Arg to His, amino acid residue 105 is ch~nged from Asp to Asn, amino acid residue lO9 is changed from Lys to Gln, and amino acid residue 111 is changed from Arg to Gln.
In another embodiment, the at least one portion of DNA
encoding pl~E protein is mutated such that amino acid residues 104, 105, 109, 111, 117, and 122 are changed.
Preferably, the at lea~t one portion of DNA is mutated such that amino acid resid~e 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue 109 is changed from Lys to Gln, amino acid residue 111 is changed from Arg to Gln, amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from Glu to Gln.
In yet another alternative embodiment, the mutation of DNA encoding pl5E protein may be effected by deleting a portion of the pl5E gene, and replacing the deleted portion of the pl5E gene, with fragment(s) or portion(s) of a gene encoding another viral protein. In one embodiment, one portion of DNA encoding the pl5E protein is replaced with a fragment of the gene encoding the p21 protein, which is an HTLV-I transmembrane protein. HTLV-I virus has been found to be resistant to binding by complement proteins and thus HTLV-I is resistant to inactivation by human serum (Hoshino, et al., Nature, Vol. 310, pgs. 324-325 ~1984)). Thus, in one embodiment, there is also provided a retroviral vector particle wherein a portion of the plSE protein has been deleted and replaced with a portion of another viral protein, such as a portion of the p21 protein.
SU~STITUTE SHEET (RULE 26) wn 93/25698 213 7 ~ 61 PCT/US93/04706 p21 protein (as shown in the ac~ompanying sequence listing) is a protein having 176 amino acid residues, and which, in relation to pl5E, has significant amino acid sequence homology. In one embodiment, at least amino acid residues 39 to 61, and lOl to 123 are deleted from pl5E
protein, and replaced with amino acid residues 34 to 56 and 96 to 118 of p21 protein. In one alternative, at least amino acid residues 39 to 123 of plSE protein are deleted and repla~ed with amino acid residues 34 to 118 of p21 protein.
In another embodiment, amino acid residues 39 to 69 of pl5E protein are deleted and replaced with amino acid residues 34 to 64 of p21 protein, and amino acid residues 96 to 123 of pl5E protein are deleted and replaced with amino acid residues 91 to 118 of p21 protein.
Vector particles thu~ generated, and which are resistant to inactivation by human serum, may be engineered surh that the vector particles may, when introduced into a patient, travel directly to a target cell or tissue. Thus, in a preferred embodiment, the vector particle further includes a protein which contains a receptor binding region that binds to a receptor of a human target cell, such as, for example, but not limited to, the amphotropic cell receptor.
The retroviral vectors hereinabove described, may be constructed by genetic engineering techniques known to those skilled in the art.
In one embodiment, the retroviral vector may be of the 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. 980-99O (1989).
In another embodiment, the retroviral vector includes a multipLe r-~triction enzyme site, or multiple cloning site.
i SUBSTiTUTE SHEET (RULE 26) W093/2~698 ; PCT/~S93/04706 i-` 2137361 The multiple cloning site includes at least four cloning, or restriction enzyme sites, wherein at least two of the sites have an average freguency of appearance in eukaryotic genes of less than once in lO,OO0 ba3e pairs; i.e., the restriction product has an average ~ize of at least 10,000 base pairs.
In general, such restriction sites, also sometimes hereinafter referred to as "rare" sites, which have an average frequency of appearance in eukaryotic genes of less than once in lO,OOO base pairs, contain a CG doublet within thsir recognition ~eguence, such doublet appearing particularly infrequently in the mammalian genome. Another measure of rarity or scarcity of a restriction enæyme site in mammals is its representation in mammalian ~iruses, such as SV40. In general, an en~yme whose recognition sequence is absent in SV40 may be a candidate for being a "rare"
mammalian cutter.
Examples of restriction enzyme sites having an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs include, but are not limited to the NotI, SnaBI, SalI, XhoI, ClaI, SacI, EagI, and SmaI
sites. Preferred cloning sites are selected from the group consisting of NotI, SnaBI, SalI, and XhoI.
Preferably, the multiple cloning site has a length no greater than about 70 base pairs, and preferably no greater than about 60 base pairs. In general, the multiple restriction enzyme site, or multiple cloning site is located between the 5'LTR and 3'LTR of the retroviral vector. The 5' end of the multiple cloning site is no greater than about 895 base pairs from the 3'~end of the 5' LTR, and preferably at least about 375 base pairs from the 3' end of the 5' LTR.
The 3' end of the multiple cloning ~ite is no greater than about 40 base pairs from ~he 5' end of the 3' LTR, and SUBSTllUTE SHEET (RULE 26) W093~25698 ~ 1 3 ~ 3 6 1 PCT~US~3/04706 f preferably at least 11 baYe pairs from the 5' end of the 3' LTR.
Such vectors may be engineered from existing retroviral vectors through genetic engineering techniques known in the art such that the resulting retroviral vector includes at least four cloning sites wherein at least two of the cloning sites are ~elected from the group consisting of the NotI, SnaBI, SalI, and XhoI cloning sites. In a preferred embodiment, the retroviral vector includes each of the NotI, SnaBI, SalI, and XhoI cloning sites.
Such a retroviral vector may serve as part of a cloning system for the transfer of genes to eukaryotic cells. Thus, there may be provided a cloning system for the manipulation of genes in a retroviral vector which încludes a retroviral vector including a multiple cloning site of the type hereinabove described, and a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, SalI, and XhoI located on the retroviral vector. The shuttle cloning vector also includes at least one desired gene which is capable of being transferred from said shuttle cloning vector to said retroviral vector.
The shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Incl~ded in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
Il SUBSrlTUTE StlEET ~'JLE 26 W093/~5698 s PCT/US93/0470~
2137~61 The shuttle cloning vector c~n be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vactor may be derived from plasmids such as pBR322; pUC18; etc.
It is also contemplated that within the scope of the present invention that the DNA encoding pl5E protein which has been mutated to render a vector particle resistant to inactivation by human serum, may be contained in an expression vehicle other than a retroviral vector. Such expression vehicles include, for example, viral vectors other ~han retroviral vectors, or any expression plasmid which is capable of being transferred into a cell l:ine which is capable of producing vector particles which include the mutated pl5E protein.
Such vectors or expression vehicles which contain DNA
encoding a mutated env protein such as the mutated pl5E
protein hereinabove described, are transferred into pre-packaging cell line to generate vector particles. In general, the pre-packaging cell line contains the gag and pol proteins of the virus, plus a retroviral vector lacking the structural gag, pol, and env proteins. An example of such a pre-packaging cell line is the GPL pre-packaging cell line which consists of an NIH 3T3 mouse fibroblast cell line , which contains an expression plasmid for MoMuLV gag-pol i protein as well as the retroviral vector LNL6 (Miller, et al., Biotechniques, Vol. 7, pgs. 980-990 (1989)). It is to be understood, however, that the scope of the present invention is not to be limited to any particular I pre-packaging cell line.
Upon transfection of the pre-packaging cell line with an expression vehicle containing DNA encoding a mutated env r Sl.~STITUTE SHEET (RULE 26) protein, the pre-packaging cell line will generate vector particles. The vector particles are then tested for complement res~stance. The vector particles which are shown to be complement resi stant (i.e., not inactivated by human serum)j therefore, contain complement resistant envelope proteins encoded by a specific envelope expression vehicle.
Such an expr~ssion vehicle can then be u~ed, 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 rontaining 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 _esistant 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 particlec 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, SUBSTIME SHEET (RULE 26) WO 93~2569~ . e - PCr/US93/04706 -` 2137~61 !
therefore, are useful for ~he introduction of desired heterologous genes into target cells ~ as a gene therapy procedure.
Thus, preferably, the vectors of the present invention ~urther include at least one heterologous gene.
Heterologous or foreign genes which may be placed into the vector or vector particles include, but are not limited to, genes which encode cytokines or cellular growth factors, such as ly~phokines, which are growth factors for lymphocytes. Other examples of foreign gen~s include, but are not limited to, genes encoding Factor VIII, Factor IX, tumor nerosis factors (TNF's), ADA, ApoE, ApoC, and Protein C.
The vectors of the present invention include one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No~ 9, pgs. 980-990 (1989), or any other promoter (eg., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and B-actin promoters). Other viral promoters which may be employed include, but are not limited to adenovirus promoters, TK
promoters, and Bl9 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The vectors of the present invention may contain regulatory elements, where necessary, to ensure tissue specific expression of the desired heterologous gene(s), and/or to regulate expression of the heterologous gene(s) in response to cellular or metabolic signals.
- Although the invention has been d~scribed with respect to retroviral vector particles, other viral vector particles ' -11-SUBSTITUTE SHEET (RULE 26J
~ ~r~ i . ' ' ' ' ' ' ' ~, (such as, for example, adenovirus and adeno-associated virus particles), or synthetic particles may be constructed wherein a region of the envelope protein in the vector particle may be mutated such that the vector particle becomes resistant to inactivation by human serum, thereby making such vector particles suitable for in vivo administration.
The invention will now be described with respect to the following example; however, the scope of the present invention is not intended to be limited thereby.
ExamPle Plasmid pCE2 was constructed from pBR322 such that the resulting plasmid pCE2 includes genes encoding the envelope proteins gp70 and pl5E. pBR322 (Figure 1) was cut with EcoRI and filled in to dèstroy the EcoRI site to give pBR3Z2 RI. pBR322 Rl was then cut with NdeI and filled in to destroy the NdeI site to give pBR322 R N.
pBR322 R N was digested with HindIII and EcoRV, and cloned into the HindIII/EcoRV fragment was a HindIII/FspI
cassette containing the gp70 and pl5E genes under the control of a cytomegalovirus (CMV) intermediate aarly promoter with a polyA (adenine) tail from SV40 (Figure 2) from plasmid pCEE. (Fi~ure 3). The HindIII/FspI cassette obtained from plasmid pCEE contains a CMV intermediate early promoter in which the BalI/SacII (bp 21 to bp 766) was converted to an HindIII~SalI fragment by linker addition;
the ecotropic envelope BglII/NheI fragment (bp 5408 to bp 7847 of MoMuLV, encoding gp70 and pl5E) was filled and EcoRI
linkers were added; and the SV40 poly A signal from BclI to BamHI (bp 2770 to bp 2533) was cloned into a BamHI site (thereby destroying the BclI site). A BglII site was added at the 3' end of the gp70 gene. (Thi~ addition does not T~TUTE SI~EET ~F~lJLE '.`6~
W093/25698 ' PCT/US93/Q4706 2137~61. -, .
alter any amino acids). The resulting plasmid is pCE2~Figure 4.~
To create the mutations in ~he pl5E gene, subcloning is carried out in a different plasmid called pUC-E2.
(Figure 6.) This plasmid is pUC18 (Figure 5) with the PvuII
fragment removed and replaced with EcoRI linker~. Such was accomplished by digesting pUC18 with PvuII to remove a 322 bp P~II fragment, and EcoRI linkers were then added. Into the EcoRI site was cloned the MoMuLV ecotropic envelope gene (i.e., the gp70 and pl5E genes from pCE2) from the BglII
site (5408) to the NheI site (7847), which ha~e been blunted and had EcoRI linkers added. The resulting pUC-E2 plasmid (Figure 6) therefore has unique BglII, SpeI, ClaI, and PvuII
sites in and around the pl5E gene.
PCR primers are then synthesized to encode the following mutations i~ the pl5E protein (using pCE2 as a template):
1. Amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 i5 changed from Glu to Gln;
BY ~nMAN 5ERUM
' ~
This invention relates to "injectable" vector particles. More particularly, this invention relates to vector particles, such as retroviral vector particles, wherein such vector particles are resistant to i~activation by human serum.
Vector particles are u~eful agents for introducing gene(~) or DNA (RNA) into a cell, such as a eukaryotic cell.
The gene(~) is controlled by an appropriate promoter.
Examples of vectors which may be employed to generate vector particles includ~ prokaryotic vectors, such as bacterial vectors; eukaryotic vectors; including fungal vectors such as yeast vectors; and viral vectors such as DNA virus vectors, RNA virus vectors, and retroviral vectors.
Retroviruses which have been employed for generating vector particles for introducing genes or DNA (RNA) into a cell include Moloney Murine Leukemia Virus, Spleen Necrosis Virus, Rous Sarcoma Virus and Harvey Sarcoma Virus. The term "introducing" as u~ed herein encompasses a variety of methods of transfering genes or DNA tRNA) into a cell. Such methods include transformation, transduction, transfection, and infection.
Vector particles have been used for introducing DNA
(RNA) into cells for gene therapy purposes. In general, such a procedure involves obtaining cells from a patient and using a vector particle to introduce desired DNA (RNA) into the cells and then providing the patient with the engineered cells for a therapeutic purpose. It would be desirable to provide alternative procedures for gene therapy. Such an alternative procedure would involve genetically engineering SUBSTITUTE SH~ET (RVLE 26) :
W093/2569~ 2 1 3 7 3 ~ 1 PC~/US93/047~6 r~
cells in vivo. In such a procedure, a vector particle which includes the desired DNA (RNA) would be administered to a patient for in v vo delivery to the cells of a patient.
It is therefore an object of the present invention to provide gene therapy by introduction of a vector particle, uch as, for example, a retroviral vector particle, into a patient, wherein the vector particle is resistant to inactivation ~y human serum.
In accordance with an aspect of the present invention, there is provided a vector particle which is resistant to inactivation by human serum. Preferably, the vector particle is a viral vector particle, and more preferably the viral vector particle is a retroviral vector particle.
The envelope portion of retroviruses include a protein known as pl5E, and Applicants have found that retroviruses are susceptible to inactivation by human serum as a result of the action of complement protein(s) present in serum on the pl5E protein portion of the retrovirus. Applicants have further found that such retroviruses can be made resistant to inactivation by human serum by mutating such pl5E
protein.
In one embodiment, there is provided a retroviral vector wherein a portion of the DNA encoding pl5E protein (shown in the accompanying sequence listing), has been mutated to render the vector particle resistant to inactivation by human serum. The terms "mutated" and "mutation" as used herein mean that the DNA encoding pl5E
protein has been changed such that at least one but not àll of the amino acids of pl5E protein have been changed (such changes can include point mutations, deletions, and/or insertions).
pl5E protein is a viral protein having 196 amino acid residues. ln viruses, sometimes all 196 amino acid residues SUBSlITUTE SHEET (RULE 26) W093/~5698 ~ ~ 3 7 ~ 6 1 PCT/US93/047~6 f~
are present, and other times, amino acid residues 181 to 196 (known as the "r" peptide), are not present, and the resulting protein is the "mature" form of pl5E known as pl2E. Thus, viruses can contain both the pl5E and pl2E
proteins. pl5E protein is anchored in the viral membrane such that amino acid residues 1 to 134 are present on the outside of the virus. Although the present invention is not to be limited to any of the following reasoning, Applicants believe complement proteins may bind to this region whereby such binding leads to inactivation and/or lysis of the retrovirus. In particular, the pl5E protein includes two regions, amino acid residues 39 to 61 (sometimes hereinafter referred to as region 1), and amino acid residues 101 to 123 (sometimes hereinafter referred to as region 2), which Applicants believe have 'an external location in the three-dimensional structure of the pl5E protein; i.e., such regions are directly exposed to human serum. Region 2 is a highly conserved region in many retroviruses, even though the amino acid ~eguences of this region are not identical in all retroviruses. Such regions are complement binding regions. Examples of complement proteins which may bind to the complement binding regions are ClS and ClQ, which bind to regions 1 and 2.
In order to inactivate the retrovirus, complement proteins bind to both region 1 and region 2. Thus, in a preferred embodiment, at least one portion of DNA encoding a complement binding region of pl5E protein has been mutated.
Such a m~ltation results in a change of at least one amino acid residue of a complement binding region of pl5E protein.
The change in at least one amino acid residue of a complement binding region of pl5E protein prevents binding of a complement protein to the complement binding region, thereby preventing complement inactivation of the SUBST~TIJTE SHEET (RULE 26~
W093/~5698 21373~1 PCT/US93/04706 retrovirus. In one embodiment, at least one amino acid residue in both complement binding region~ of pl5E protein is changed, whereas in another embodiment, at least one amino acid residue in one of the complement binding regions i 9 cha~ged.
It is to be understood, howaver, that the entire DNA
se-guence encoding pl5E protein cannot be mutated because such a change renders the vectors unsuitable for in vivo use.
In one embodiment, the at least one portion of DNA
encoding pl5E protein is mutated such that at least one positively charged amino acid residue or negatively charged amino acid residue is changed to an amino acild residue having the opposite charge.
The positively charged amino acids are His, Lys, and Arg.
The negatively charged amino acids are Asp and Glu.
In another~embodiment, the at least one portion of DNA
encoding pl~E protein is mutated such that at least one positively charged amino acid or negatively charged amino acid is changed to a noncharged amino acid.
In one embodiment, the at least one portion of DNA
encoding a complement binding region of pl5~ protein, which is mutatad, encodes one or more of amino acid residues 101 to 123 of pl5E protein. In one em~odiment, the at least one portion of DNA encoding pl5E protein is mutated such that amino acid residue 122 is changed.
In one embodiment, the at least one portion of DNA
encoding pl5E protein is mutated such that at least one of amino acid residues 117~ and 122 are changed. Preferably, amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 i8 changed from Glu to Gln.
SUBSTITUTE SHEET (RULE 26) W093/25698 ~ PCT/US93/04706 In another embodiment, the at least one portion of DNA
encoding pl5~ protein is mutated ~uch that amino acid residues 104, 105, 109, and 111 are changed. Preferably, amino acid residue 104 is changed from Arg to His, amino acid residue 105 is ch~nged from Asp to Asn, amino acid residue lO9 is changed from Lys to Gln, and amino acid residue 111 is changed from Arg to Gln.
In another embodiment, the at least one portion of DNA
encoding pl~E protein is mutated such that amino acid residues 104, 105, 109, 111, 117, and 122 are changed.
Preferably, the at lea~t one portion of DNA is mutated such that amino acid resid~e 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue 109 is changed from Lys to Gln, amino acid residue 111 is changed from Arg to Gln, amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from Glu to Gln.
In yet another alternative embodiment, the mutation of DNA encoding pl5E protein may be effected by deleting a portion of the pl5E gene, and replacing the deleted portion of the pl5E gene, with fragment(s) or portion(s) of a gene encoding another viral protein. In one embodiment, one portion of DNA encoding the pl5E protein is replaced with a fragment of the gene encoding the p21 protein, which is an HTLV-I transmembrane protein. HTLV-I virus has been found to be resistant to binding by complement proteins and thus HTLV-I is resistant to inactivation by human serum (Hoshino, et al., Nature, Vol. 310, pgs. 324-325 ~1984)). Thus, in one embodiment, there is also provided a retroviral vector particle wherein a portion of the plSE protein has been deleted and replaced with a portion of another viral protein, such as a portion of the p21 protein.
SU~STITUTE SHEET (RULE 26) wn 93/25698 213 7 ~ 61 PCT/US93/04706 p21 protein (as shown in the ac~ompanying sequence listing) is a protein having 176 amino acid residues, and which, in relation to pl5E, has significant amino acid sequence homology. In one embodiment, at least amino acid residues 39 to 61, and lOl to 123 are deleted from pl5E
protein, and replaced with amino acid residues 34 to 56 and 96 to 118 of p21 protein. In one alternative, at least amino acid residues 39 to 123 of plSE protein are deleted and repla~ed with amino acid residues 34 to 118 of p21 protein.
In another embodiment, amino acid residues 39 to 69 of pl5E protein are deleted and replaced with amino acid residues 34 to 64 of p21 protein, and amino acid residues 96 to 123 of pl5E protein are deleted and replaced with amino acid residues 91 to 118 of p21 protein.
Vector particles thu~ generated, and which are resistant to inactivation by human serum, may be engineered surh that the vector particles may, when introduced into a patient, travel directly to a target cell or tissue. Thus, in a preferred embodiment, the vector particle further includes a protein which contains a receptor binding region that binds to a receptor of a human target cell, such as, for example, but not limited to, the amphotropic cell receptor.
The retroviral vectors hereinabove described, may be constructed by genetic engineering techniques known to those skilled in the art.
In one embodiment, the retroviral vector may be of the 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. 980-99O (1989).
In another embodiment, the retroviral vector includes a multipLe r-~triction enzyme site, or multiple cloning site.
i SUBSTiTUTE SHEET (RULE 26) W093/2~698 ; PCT/~S93/04706 i-` 2137361 The multiple cloning site includes at least four cloning, or restriction enzyme sites, wherein at least two of the sites have an average freguency of appearance in eukaryotic genes of less than once in lO,OO0 ba3e pairs; i.e., the restriction product has an average ~ize of at least 10,000 base pairs.
In general, such restriction sites, also sometimes hereinafter referred to as "rare" sites, which have an average frequency of appearance in eukaryotic genes of less than once in lO,OOO base pairs, contain a CG doublet within thsir recognition ~eguence, such doublet appearing particularly infrequently in the mammalian genome. Another measure of rarity or scarcity of a restriction enæyme site in mammals is its representation in mammalian ~iruses, such as SV40. In general, an en~yme whose recognition sequence is absent in SV40 may be a candidate for being a "rare"
mammalian cutter.
Examples of restriction enzyme sites having an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs include, but are not limited to the NotI, SnaBI, SalI, XhoI, ClaI, SacI, EagI, and SmaI
sites. Preferred cloning sites are selected from the group consisting of NotI, SnaBI, SalI, and XhoI.
Preferably, the multiple cloning site has a length no greater than about 70 base pairs, and preferably no greater than about 60 base pairs. In general, the multiple restriction enzyme site, or multiple cloning site is located between the 5'LTR and 3'LTR of the retroviral vector. The 5' end of the multiple cloning site is no greater than about 895 base pairs from the 3'~end of the 5' LTR, and preferably at least about 375 base pairs from the 3' end of the 5' LTR.
The 3' end of the multiple cloning ~ite is no greater than about 40 base pairs from ~he 5' end of the 3' LTR, and SUBSTllUTE SHEET (RULE 26) W093~25698 ~ 1 3 ~ 3 6 1 PCT~US~3/04706 f preferably at least 11 baYe pairs from the 5' end of the 3' LTR.
Such vectors may be engineered from existing retroviral vectors through genetic engineering techniques known in the art such that the resulting retroviral vector includes at least four cloning sites wherein at least two of the cloning sites are ~elected from the group consisting of the NotI, SnaBI, SalI, and XhoI cloning sites. In a preferred embodiment, the retroviral vector includes each of the NotI, SnaBI, SalI, and XhoI cloning sites.
Such a retroviral vector may serve as part of a cloning system for the transfer of genes to eukaryotic cells. Thus, there may be provided a cloning system for the manipulation of genes in a retroviral vector which încludes a retroviral vector including a multiple cloning site of the type hereinabove described, and a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, SalI, and XhoI located on the retroviral vector. The shuttle cloning vector also includes at least one desired gene which is capable of being transferred from said shuttle cloning vector to said retroviral vector.
The shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Incl~ded in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
Il SUBSrlTUTE StlEET ~'JLE 26 W093/~5698 s PCT/US93/0470~
2137~61 The shuttle cloning vector c~n be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vactor may be derived from plasmids such as pBR322; pUC18; etc.
It is also contemplated that within the scope of the present invention that the DNA encoding pl5E protein which has been mutated to render a vector particle resistant to inactivation by human serum, may be contained in an expression vehicle other than a retroviral vector. Such expression vehicles include, for example, viral vectors other ~han retroviral vectors, or any expression plasmid which is capable of being transferred into a cell l:ine which is capable of producing vector particles which include the mutated pl5E protein.
Such vectors or expression vehicles which contain DNA
encoding a mutated env protein such as the mutated pl5E
protein hereinabove described, are transferred into pre-packaging cell line to generate vector particles. In general, the pre-packaging cell line contains the gag and pol proteins of the virus, plus a retroviral vector lacking the structural gag, pol, and env proteins. An example of such a pre-packaging cell line is the GPL pre-packaging cell line which consists of an NIH 3T3 mouse fibroblast cell line , which contains an expression plasmid for MoMuLV gag-pol i protein as well as the retroviral vector LNL6 (Miller, et al., Biotechniques, Vol. 7, pgs. 980-990 (1989)). It is to be understood, however, that the scope of the present invention is not to be limited to any particular I pre-packaging cell line.
Upon transfection of the pre-packaging cell line with an expression vehicle containing DNA encoding a mutated env r Sl.~STITUTE SHEET (RULE 26) protein, the pre-packaging cell line will generate vector particles. The vector particles are then tested for complement res~stance. The vector particles which are shown to be complement resi stant (i.e., not inactivated by human serum)j therefore, contain complement resistant envelope proteins encoded by a specific envelope expression vehicle.
Such an expr~ssion vehicle can then be u~ed, 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 rontaining 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 _esistant 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 particlec 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, SUBSTIME SHEET (RULE 26) WO 93~2569~ . e - PCr/US93/04706 -` 2137~61 !
therefore, are useful for ~he introduction of desired heterologous genes into target cells ~ as a gene therapy procedure.
Thus, preferably, the vectors of the present invention ~urther include at least one heterologous gene.
Heterologous or foreign genes which may be placed into the vector or vector particles include, but are not limited to, genes which encode cytokines or cellular growth factors, such as ly~phokines, which are growth factors for lymphocytes. Other examples of foreign gen~s include, but are not limited to, genes encoding Factor VIII, Factor IX, tumor nerosis factors (TNF's), ADA, ApoE, ApoC, and Protein C.
The vectors of the present invention include one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No~ 9, pgs. 980-990 (1989), or any other promoter (eg., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and B-actin promoters). Other viral promoters which may be employed include, but are not limited to adenovirus promoters, TK
promoters, and Bl9 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The vectors of the present invention may contain regulatory elements, where necessary, to ensure tissue specific expression of the desired heterologous gene(s), and/or to regulate expression of the heterologous gene(s) in response to cellular or metabolic signals.
- Although the invention has been d~scribed with respect to retroviral vector particles, other viral vector particles ' -11-SUBSTITUTE SHEET (RULE 26J
~ ~r~ i . ' ' ' ' ' ' ' ~, (such as, for example, adenovirus and adeno-associated virus particles), or synthetic particles may be constructed wherein a region of the envelope protein in the vector particle may be mutated such that the vector particle becomes resistant to inactivation by human serum, thereby making such vector particles suitable for in vivo administration.
The invention will now be described with respect to the following example; however, the scope of the present invention is not intended to be limited thereby.
ExamPle Plasmid pCE2 was constructed from pBR322 such that the resulting plasmid pCE2 includes genes encoding the envelope proteins gp70 and pl5E. pBR322 (Figure 1) was cut with EcoRI and filled in to dèstroy the EcoRI site to give pBR3Z2 RI. pBR322 Rl was then cut with NdeI and filled in to destroy the NdeI site to give pBR322 R N.
pBR322 R N was digested with HindIII and EcoRV, and cloned into the HindIII/EcoRV fragment was a HindIII/FspI
cassette containing the gp70 and pl5E genes under the control of a cytomegalovirus (CMV) intermediate aarly promoter with a polyA (adenine) tail from SV40 (Figure 2) from plasmid pCEE. (Fi~ure 3). The HindIII/FspI cassette obtained from plasmid pCEE contains a CMV intermediate early promoter in which the BalI/SacII (bp 21 to bp 766) was converted to an HindIII~SalI fragment by linker addition;
the ecotropic envelope BglII/NheI fragment (bp 5408 to bp 7847 of MoMuLV, encoding gp70 and pl5E) was filled and EcoRI
linkers were added; and the SV40 poly A signal from BclI to BamHI (bp 2770 to bp 2533) was cloned into a BamHI site (thereby destroying the BclI site). A BglII site was added at the 3' end of the gp70 gene. (Thi~ addition does not T~TUTE SI~EET ~F~lJLE '.`6~
W093/25698 ' PCT/US93/Q4706 2137~61. -, .
alter any amino acids). The resulting plasmid is pCE2~Figure 4.~
To create the mutations in ~he pl5E gene, subcloning is carried out in a different plasmid called pUC-E2.
(Figure 6.) This plasmid is pUC18 (Figure 5) with the PvuII
fragment removed and replaced with EcoRI linker~. Such was accomplished by digesting pUC18 with PvuII to remove a 322 bp P~II fragment, and EcoRI linkers were then added. Into the EcoRI site was cloned the MoMuLV ecotropic envelope gene (i.e., the gp70 and pl5E genes from pCE2) from the BglII
site (5408) to the NheI site (7847), which ha~e been blunted and had EcoRI linkers added. The resulting pUC-E2 plasmid (Figure 6) therefore has unique BglII, SpeI, ClaI, and PvuII
sites in and around the pl5E gene.
PCR primers are then synthesized to encode the following mutations i~ the pl5E protein (using pCE2 as a template):
1. Amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 i5 changed from Glu to Gln;
2. Amino acid residue 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue 109 is changed from Lys to Gln, and amino acid residue 111 is changed from Arg to Gln; and 3. Amino acid residue 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue 109 is changed from Lys to Gln, amino acid residue 111 is changed from Arg to Gln, amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from Glu to Gln.
Each PCR prod~ct is digested with SpeI and PvuII, and cloned into pUC-E2 at the unique SpeI and PvuII sites. The resulting plasmids are then seguenced to confirm the point mutations.
SU~S~ITUTE SHEt, ~RllLE ~o`
W093/2~698 ~1 3 7 3 6 1 PCT/US93/04706 f ~
_, DNA fra~ments bearing these mutations are then subcloned into the expression plasmid pCE2. pCEZ is digested with EcoRI and the envelope DNA fragment is removed and replaced with the EcoRI envelope fragment ~rom the pUC-E2 plasmids. The resulting pCE2 plasmids are then checked for orientation of the EcoRI fragment and seguenced again (only at the cloning site junctions and at the regions bearing the point mutations) to confirm the presence of the newly created mutated pl5E genes. The resulting expression plasmids are identified as follows:
pCR68 - includes mutations in which amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from Glu to Gln;
pCR69 - includes mutations in which amino acid residue 104 is changad from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue lO9 is changed from Lys to Gln, and amino acid residue 111 is changed from Arg to Gln; and pCR70 - includes mutations in which amino acid residue 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue lO9 is changed from Lys to Gln, amino acid residue 111 is changed from Arg to Gln, amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from &lu to Gln.
Plasmids pCR68, pCR69, and pCR70 are transfected separately into the GPL pre-packaging cell line. The GPL
pre-packaging cell line consists of an NIH 3T3 mouse fibroblast cell line which contains an expression plasmid for MoMuLV gag-pol protein as well as the retroviral vector LNL6 (Miller, et al., 1989). Upon transfection with pCR68, pCR69, or pCR70, the GPL packaging cell line produces vector paxticles.
r.~S~ ITE SHE~ (RULE ~
W~93/2S698 ~ PCT/US93/04706 ; 2~3736~
Transiently expressed vector particles are collected with cell supernatant at 48-72 hrs. post-transfection.
The vector particles generated as hereinabove described may then be assayed for vector titer by techniques known to those ~killed in the art. The vector particles may also be collected in viral supernatant and concentrated, if necéssary, according to procedures Xnown to those skilled in the art in order to employ such vector particles in assays or in therapeutic procedures.
Advantages of the present invention include the ability to introduce vector particles directly into a human patient whereby the vector particle is not lysed or inactivated by human serum upon such introduction. Thus, the vector particles of the present invention enable one to deliver desired genes to a patient in vivo. Such vector particles may also be engineered such that they are "targetable", as well as injectable, thereby enabling the vector particles to travel directly to a target cell or tissue without being lysed or inactivated by human serum.
It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.
~ TUT SHE~ (~J! ~ ~) W0~3/2$698 2 1 3 ~ 3 6 i PCT/US93/04706 ~ . ' SEQUENCE LISTING
51~ GE~ERAL INFORMATION:
(i) APPLICANT: Anderson, W. French Mason, Jam~s M.
(ii) TITLE OF INVENTION: Vector Particles Resistant to Inactivation - by Human Serum (iii) NUMBER QF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) ~DDRESSEE: Carella, Byrne, Bain, Gilfillan, Cecchi & St:ewart (B) STREET: 6 Becker Farm Road (C) CITY: Ro~eland (~) STATE: N~w Jersey (E) COUNTRY: ' USA
(F) ZIP: 07068 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch diskette (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: DW4.V2 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILIN~ DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATI~N:
: (A) NAME: Olstein, Elliot M.
(B) REGISTRATION NUMBER: 24,025 (C) REFERENCE/DOCKET NUMBER: 271010-73 (ix) TELECOMMUNICATION INFORMATION:
IT~TE SHE~ ~RU~ _ ?6' WO 93/25698 ~ 1 3 7 3 6 1 PCr/US93/0 (A) TELEP~IONE: 201-994-1700 (B~ TELEFA~C: 201-994-1744 ( 2 ) INFORMATION FOR SEQ ID NO ~
t ~: ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 196 amino acids B ) TYP~5 ~ amino acid ~ C ) STRANDEDNESS:
( D ) TOPOLOG~Y: linear ( ii ) MOLECULE TYPE: protein ( ix ) FEATURE:
(A) NAME/KEY: pl5E protein.
(xi ) SEQUENCE; DESCRIPTION: SEQ ID NO: 1:
Giu Pro Val Ser Leu Thr Leu Al~ Leu Leu ` 5 10 Leu C;ly Gly Leu Thr Met Gly Gly I le Ala Ala Gly I le Gly Thr Gly Thr Thr Ala Leu Met Ala Thr Gln Gln Phe Gln Gln Leu Gln Ala Ala Val Gln Asp Aæp Leu Arg Glu Val Glu Lys Ser I le Ser Asn Leu Glu Lys Ser ~5 60 Leu Thr Ser Leu Ser Glu Val Val Leu Gln Asn Ar~ Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ala Asp His Thr q ~ -17-,: . . ~ , .
UTE SHEET ~RU!F ~
W093t2569$ 2 1 ~ 7 3 ~ i P~/USg3/04706 Gly Leu Val Arg Asp Ser Met Ala Lys Leu Arg Glu Arg Leu Asn Gln Arg Gln Lys Leu Phe Glu Ser Thr Gln Gly Trp Phe Glu Gly Leu Phe Asn Arg Ser Pro Trp Phe Thr Thr Leu Ile Ser Thr Ile Met Gly Pro Leu Ile Val Leu Leu Met Ile Leu Leu Phe Gly Pro 155 l~iO
Cys Ile Leu Asn Arg Leu Val Gln Phe Val 165 1'70 Lys Asp Arg Ile Ser Val Val Gln Ala Leu 175 1~0 Val Leu Thr Gln Gln Tyr His Gln Leu Lys 1~5 190 Pro Ile Glu Tyr Glu Pro (2) INFO~MATION EOR SEQ ID NO:2:
~ (i) SEQUENCE CH~RACTERISTICS:
i (A~ LENGTH: 176 amino acids (B) TYPE: amino acid (C) 5TRANDEDNESS:
(D) TOPOLOGY: linear , l (ii) MOLECULE TYPE: protein : .
. (ix) FEATURE
. (A) NAME/KEY: p21 protein : ; ~
~ -18-.~
TUT~ SHEFr !R~IL~ r.
, .
WO 93/25~98 ~ ' PCT/US93/04706 2~37361 ` -(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:2:
Ala Val Pro Val Ala Val Trp Leu Val Ser Ala Leu Ala Met Gly Ala Gly Val Ala Gly 15 20 ;
Arg Ile Thr Gly Ser Met Ser Leu Ala Ser ~5 30 Gly Lys Ser Leu Leu His Glu Val Asp Lys Asp Ile Ser Gln Leu Thr Gln Ala Ile Val Lys Asn His Lys A~n Leu Leu 1ys Ile Ala Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu ' 65 70 Asp Leu Leu Phe Trp Glu Gln Gly Gly Leu Cys Lys ~la Leu Gln Glu Gln Cys Cys Phe Leu Asn Ile Thr Asn Ser Hi s Val Ser I le ~ Leu Gln Glu Arg Pro Pro Leu Glu Asn Arg i 105 llO
Val Leu Thr Gly Trp Gly Leu Rsn Trp Asp ~- 115 120 Leu Gly Leu Ser Gln Trp Ala Arg Glu Ala ` 125 130 Leu Gln Thr Gly Ile Thr Leu Val Ala Leu Leu Leu Leu Val Ile Leu Ala Gly Pro Cys ~ 145 150 ;. Ile Leu Arg Gln Leu Arg His Leu Pro Ser : 155 160 c ~i . -19-"i ,, ", SUBS~ITUTE SHEET ~RVLE 26) r~
WO 93~25698 2 ~ ~ 7 3 6 i P~/US93/04706 Arg Val Arg Tyr Pro Hi 5 Tyr Ser Leu I le Asn Pro Glu Ser Ser Leu , i r -~0-5U~ IUTESHE~RU~
Each PCR prod~ct is digested with SpeI and PvuII, and cloned into pUC-E2 at the unique SpeI and PvuII sites. The resulting plasmids are then seguenced to confirm the point mutations.
SU~S~ITUTE SHEt, ~RllLE ~o`
W093/2~698 ~1 3 7 3 6 1 PCT/US93/04706 f ~
_, DNA fra~ments bearing these mutations are then subcloned into the expression plasmid pCE2. pCEZ is digested with EcoRI and the envelope DNA fragment is removed and replaced with the EcoRI envelope fragment ~rom the pUC-E2 plasmids. The resulting pCE2 plasmids are then checked for orientation of the EcoRI fragment and seguenced again (only at the cloning site junctions and at the regions bearing the point mutations) to confirm the presence of the newly created mutated pl5E genes. The resulting expression plasmids are identified as follows:
pCR68 - includes mutations in which amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from Glu to Gln;
pCR69 - includes mutations in which amino acid residue 104 is changad from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue lO9 is changed from Lys to Gln, and amino acid residue 111 is changed from Arg to Gln; and pCR70 - includes mutations in which amino acid residue 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue lO9 is changed from Lys to Gln, amino acid residue 111 is changed from Arg to Gln, amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from &lu to Gln.
Plasmids pCR68, pCR69, and pCR70 are transfected separately into the GPL pre-packaging cell line. The GPL
pre-packaging cell line consists of an NIH 3T3 mouse fibroblast cell line which contains an expression plasmid for MoMuLV gag-pol protein as well as the retroviral vector LNL6 (Miller, et al., 1989). Upon transfection with pCR68, pCR69, or pCR70, the GPL packaging cell line produces vector paxticles.
r.~S~ ITE SHE~ (RULE ~
W~93/2S698 ~ PCT/US93/04706 ; 2~3736~
Transiently expressed vector particles are collected with cell supernatant at 48-72 hrs. post-transfection.
The vector particles generated as hereinabove described may then be assayed for vector titer by techniques known to those ~killed in the art. The vector particles may also be collected in viral supernatant and concentrated, if necéssary, according to procedures Xnown to those skilled in the art in order to employ such vector particles in assays or in therapeutic procedures.
Advantages of the present invention include the ability to introduce vector particles directly into a human patient whereby the vector particle is not lysed or inactivated by human serum upon such introduction. Thus, the vector particles of the present invention enable one to deliver desired genes to a patient in vivo. Such vector particles may also be engineered such that they are "targetable", as well as injectable, thereby enabling the vector particles to travel directly to a target cell or tissue without being lysed or inactivated by human serum.
It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.
~ TUT SHE~ (~J! ~ ~) W0~3/2$698 2 1 3 ~ 3 6 i PCT/US93/04706 ~ . ' SEQUENCE LISTING
51~ GE~ERAL INFORMATION:
(i) APPLICANT: Anderson, W. French Mason, Jam~s M.
(ii) TITLE OF INVENTION: Vector Particles Resistant to Inactivation - by Human Serum (iii) NUMBER QF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) ~DDRESSEE: Carella, Byrne, Bain, Gilfillan, Cecchi & St:ewart (B) STREET: 6 Becker Farm Road (C) CITY: Ro~eland (~) STATE: N~w Jersey (E) COUNTRY: ' USA
(F) ZIP: 07068 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch diskette (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: DW4.V2 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILIN~ DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATI~N:
: (A) NAME: Olstein, Elliot M.
(B) REGISTRATION NUMBER: 24,025 (C) REFERENCE/DOCKET NUMBER: 271010-73 (ix) TELECOMMUNICATION INFORMATION:
IT~TE SHE~ ~RU~ _ ?6' WO 93/25698 ~ 1 3 7 3 6 1 PCr/US93/0 (A) TELEP~IONE: 201-994-1700 (B~ TELEFA~C: 201-994-1744 ( 2 ) INFORMATION FOR SEQ ID NO ~
t ~: ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 196 amino acids B ) TYP~5 ~ amino acid ~ C ) STRANDEDNESS:
( D ) TOPOLOG~Y: linear ( ii ) MOLECULE TYPE: protein ( ix ) FEATURE:
(A) NAME/KEY: pl5E protein.
(xi ) SEQUENCE; DESCRIPTION: SEQ ID NO: 1:
Giu Pro Val Ser Leu Thr Leu Al~ Leu Leu ` 5 10 Leu C;ly Gly Leu Thr Met Gly Gly I le Ala Ala Gly I le Gly Thr Gly Thr Thr Ala Leu Met Ala Thr Gln Gln Phe Gln Gln Leu Gln Ala Ala Val Gln Asp Aæp Leu Arg Glu Val Glu Lys Ser I le Ser Asn Leu Glu Lys Ser ~5 60 Leu Thr Ser Leu Ser Glu Val Val Leu Gln Asn Ar~ Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ala Asp His Thr q ~ -17-,: . . ~ , .
UTE SHEET ~RU!F ~
W093t2569$ 2 1 ~ 7 3 ~ i P~/USg3/04706 Gly Leu Val Arg Asp Ser Met Ala Lys Leu Arg Glu Arg Leu Asn Gln Arg Gln Lys Leu Phe Glu Ser Thr Gln Gly Trp Phe Glu Gly Leu Phe Asn Arg Ser Pro Trp Phe Thr Thr Leu Ile Ser Thr Ile Met Gly Pro Leu Ile Val Leu Leu Met Ile Leu Leu Phe Gly Pro 155 l~iO
Cys Ile Leu Asn Arg Leu Val Gln Phe Val 165 1'70 Lys Asp Arg Ile Ser Val Val Gln Ala Leu 175 1~0 Val Leu Thr Gln Gln Tyr His Gln Leu Lys 1~5 190 Pro Ile Glu Tyr Glu Pro (2) INFO~MATION EOR SEQ ID NO:2:
~ (i) SEQUENCE CH~RACTERISTICS:
i (A~ LENGTH: 176 amino acids (B) TYPE: amino acid (C) 5TRANDEDNESS:
(D) TOPOLOGY: linear , l (ii) MOLECULE TYPE: protein : .
. (ix) FEATURE
. (A) NAME/KEY: p21 protein : ; ~
~ -18-.~
TUT~ SHEFr !R~IL~ r.
, .
WO 93/25~98 ~ ' PCT/US93/04706 2~37361 ` -(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:2:
Ala Val Pro Val Ala Val Trp Leu Val Ser Ala Leu Ala Met Gly Ala Gly Val Ala Gly 15 20 ;
Arg Ile Thr Gly Ser Met Ser Leu Ala Ser ~5 30 Gly Lys Ser Leu Leu His Glu Val Asp Lys Asp Ile Ser Gln Leu Thr Gln Ala Ile Val Lys Asn His Lys A~n Leu Leu 1ys Ile Ala Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu ' 65 70 Asp Leu Leu Phe Trp Glu Gln Gly Gly Leu Cys Lys ~la Leu Gln Glu Gln Cys Cys Phe Leu Asn Ile Thr Asn Ser Hi s Val Ser I le ~ Leu Gln Glu Arg Pro Pro Leu Glu Asn Arg i 105 llO
Val Leu Thr Gly Trp Gly Leu Rsn Trp Asp ~- 115 120 Leu Gly Leu Ser Gln Trp Ala Arg Glu Ala ` 125 130 Leu Gln Thr Gly Ile Thr Leu Val Ala Leu Leu Leu Leu Val Ile Leu Ala Gly Pro Cys ~ 145 150 ;. Ile Leu Arg Gln Leu Arg His Leu Pro Ser : 155 160 c ~i . -19-"i ,, ", SUBS~ITUTE SHEET ~RVLE 26) r~
WO 93~25698 2 ~ ~ 7 3 6 i P~/US93/04706 Arg Val Arg Tyr Pro Hi 5 Tyr Ser Leu I le Asn Pro Glu Ser Ser Leu , i r -~0-5U~ IUTESHE~RU~
Claims (21)
1. A retroviral vector particle, said vector particle being resistant to inactivation by human serum.
2. The vector particle of Claim 1 wherein said vector particle includes p15E protein, and wherein a portion but not all of the p15E protein has been mutated to render the vector particles resistant to inactivation by human serum.
3. The vector particle of Claim 2 wherein a portion of the p15E protein has been mutated such that at least one amino acid of p15E protein has been changed.
4. The vector particle of Claim 3 wherein at least one portion of a complement binding region of p15E protein has been mutated.
5. The vector particle of Claim 4 wherein said complement binding region is selected from the group consisting of amino acid residues 39 to 61 and 101 to 123 of p15E protein.
6. The vector particle of Claim 5 wherein said complement binding region is amino acid residues 101 to 123 of p15E protein.
7. The vector particle of Claim 6 wherein p15E
protein is mutated such that amino acid residue 122 of p15E
protein is changed.
protein is mutated such that amino acid residue 122 of p15E
protein is changed.
8. The vector particle of Claim 6 wherein p15E
protein is mutated such that amino acid residues 117 and 122 are changed.
protein is mutated such that amino acid residues 117 and 122 are changed.
9. The vector particle of Claim 8 wherein p15E
protein is mutated such that amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from Glu to Gln.
protein is mutated such that amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from Glu to Gln.
10. The vector particle of Claim 6 wherein p15E
protein is mutated such that amino acid residues 104, 105, 109, and 111 are changed.
protein is mutated such that amino acid residues 104, 105, 109, and 111 are changed.
11. The vector particle of Claim 10 wherein p15E
protein is mutated such that amino acid residue 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue 109 is changed from Lys to Gln, and amino acid residue 111 is changed from Arg to Gln.
protein is mutated such that amino acid residue 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue 109 is changed from Lys to Gln, and amino acid residue 111 is changed from Arg to Gln.
12. The vector particle of Claim 6 wherein p15E
protein is mutated such that amino acid residues 104, 105, 109, 111, 117, and 122 are changed.
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 p15E
protein is mutated such that amino acid residue 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue 109 is changed from Lys to Gln, amino acid residue 111 is changed from Arg to Gln, amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from Glu to Gln.
protein is mutated such that amino acid residue 104 is changed from Arg to His, amino acid residue 105 is changed from Asp to Asn, amino acid residue 109 is changed from Lys to Gln, amino acid residue 111 is changed from Arg to Gln, amino acid residue 117 is changed from Arg to Glu, and amino acid residue 122 is changed from Glu to Gln.
14. The vector particle of Claim 5 wherein p15E
protein is mutated such that at least one positively or negatively charged amino acid is changed to an amino acid having the opposite charge.
protein is mutated such that at least one positively or negatively charged amino acid is changed to an amino acid having the opposite charge.
15. The vector particle of Claim 5 wherein p15E
protein is mutated such that at least one positively charged or negatively charged amino acid is changed to a non-charged amino acid.
protein is mutated such that at least one positively charged or negatively charged amino acid is changed to a non-charged amino acid.
16. The vector particle of Claim 1 wherein said vector particle further includes a protein containing a receptor binding region that binds to a receptor of a human target cell.
17. The vector particle of Claim 16 and further containing a heterologous gene.
18. Eukaryotic cells transformed with the vector particles of Claim 17.
19. An expression vehicle including DNA encoding p15E protein, wherein said DNA encoding p15E protein is mutated such that when said mutated p15E protein encoded by said DNA is included in a vector particle, said vector particle is resistant to inactivation by human serum.
20. A packaging cell line including the expression vehicle of Claim 19.
21. A viral vector particle, said viral vector particle being resistant to inactivation by human serum.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US89660392A | 1992-06-10 | 1992-06-10 | |
| US896,603 | 1992-06-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2137361A1 true CA2137361A1 (en) | 1993-12-23 |
Family
ID=25406489
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2137361 Abandoned CA2137361A1 (en) | 1992-06-10 | 1993-05-14 | Vector particles resistant to inactivation by human serum |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0644946A4 (en) |
| JP (1) | JPH09507741A (en) |
| CA (1) | CA2137361A1 (en) |
| WO (1) | WO1993025698A1 (en) |
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| WO1991019798A1 (en) * | 1990-06-20 | 1991-12-26 | Dana Farber Cancer Institute | Vectors containing hiv packaging sequences, packaging defective hiv vectors, and uses thereof |
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- 1993-05-14 WO PCT/US1993/004706 patent/WO1993025698A1/en not_active Application Discontinuation
- 1993-05-14 CA CA 2137361 patent/CA2137361A1/en not_active Abandoned
- 1993-05-14 EP EP93913964A patent/EP0644946A4/en not_active Withdrawn
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| WO1993025698A1 (en) | 1993-12-23 |
| EP0644946A4 (en) | 1997-03-12 |
| EP0644946A1 (en) | 1995-03-29 |
| JPH09507741A (en) | 1997-08-12 |
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