WO2000002909A2 - Amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins - Google Patents
Amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins Download PDFInfo
- Publication number
- WO2000002909A2 WO2000002909A2 PCT/IB1999/001261 IB9901261W WO0002909A2 WO 2000002909 A2 WO2000002909 A2 WO 2000002909A2 IB 9901261 W IB9901261 W IB 9901261W WO 0002909 A2 WO0002909 A2 WO 0002909A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- membrane
- envelope protein
- viral
- retroviral
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13041—Use of virus, viral particle or viral elements as a vector
- C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13041—Use of virus, viral particle or viral elements as a vector
- C12N2740/13045—Special targeting system for viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
Definitions
- This invention relates to isolated amphiphilic peptides that are derived from the cytoplasmic tail and / or the membrane- spanning region of viral envelope proteins and, in particular, the cytoplasmic tail region of the transmembrane subunit of retroviral envelope proteins, and to derivatives or analogues of such peptides that maintain the amphiphilic structure of such peptides, which provides their membrane destabilization activity.
- This invention also relates to modified enveloped viruses in which the viral envelope is modified to include the foregoing peptides or their derivatives or analogues.
- the present invention relates to retroviruses having modified envelope proteins, wherein the peptides or their derivatives or analogues are included in the surface (SU) subunit and/or the external region of the transmembrane (TM) subunit of the retroviral envelope protein, or are attached to the exterior and/or interior of the retroviral membrane, independently of and in addition to, or in lieu of, the viral envelope protein.
- modified enveloped viruses also may include a targeting peptide containing a binding region that binds to a ligand.
- Retroviruses in general include a "core” that contains a retroviral genome, nucleoprotein, protease, reverse transcriptase, and integrase enclosed within a capsid.
- a retroviral envelope surrounds the capsid.
- the retroviral envelope includes a viral membrane and viral envelope protein.
- the retroviral envelope protein is a post-translationally cleaved heterodimer of a surface subunit (SU) and a transmembrane subunit (TM) .
- the TM includes an external region which is on the external side of the viral membrane and is complexed or associated with the SU; a membrane-spanning region, which is located within the viral membrane; and a cytoplasmic tail region, which is on the internal side of the viral membrane.
- the retroviral envelope protein (Env) is functional at two key steps in host or target cell entry: 1) binding of the cellular receptor and 2) fusion with the cellular membrane.
- the initial steps in viral entry are understood in considerable detail (White, Science, Vol. 258, pgs. 917-924 (1992)).
- the first interaction between a retrovirus and a host or target cell occurs as the SU binds to a receptor on the cell.
- the TM undergoes a major conformational change during which its N-terminal end, known as the "fusion peptide,” is liberated from its hydrophobic environment within the SU and inserts into the host or target cell membrane.
- Peptides representing the portion of the TM immediately adjacent to the fusion peptide have the propensity to separate into monomers within the host cell membrane. (Yu, et . al..Science, Vol., 266, pgs . 274-276 (1994)). Such an event may initiate juxtaposition of the viral and host or target cell membranes .
- the viral envelope protein-cell receptor interaction is followed by multiple receptor recruitment, which is speculated to assist in the merging of the two membranes (Melikyan, et al . , J . Cell. Biol. , Vol. 13, pgs. 679-691 (1995)).
- the current literature provides evidence that, whereas a viral envelope protein is necessary and sufficient to induce full fusion (Jones, et al . , J. Virol . , Vol, 67 pgs. 67-74 (1993)), an envelope protein ectodomain (i.e.
- the external region) attached to a membrane by a glycolipid linker anchor induces fusion of only the outer lipid bilayers (i.e., hemifusion) , but does not cause complete fusion (Kemble, et al., Cell, Vol.76, pgs. 383-391 (1994)).
- Such data provide speculation that the membrane-spanning region and/or cytoplasmic tail region of the TM may be required for bringing envelope protein mediated fusion to completion.
- the present invention is directed to a cytoplasmic tail domain or region of the envelope protein that may lower the kinetic barrier to membrane fusion.
- the present invention is directed to isolated peptides including an amino acid sequence having an amphiphilic structure.
- the amino acid sequence may be derived from a viral envelope protein and, in particular, a retroviral envelope protein.
- Such amino acid sequence includes at least a portion of the amino acid sequence present in the cytoplasmic tail region of the TM of the envelope protein adjacent to the membrane-spanning region of the TM of the envelope protein.
- the peptide may or may not include at least a portion of the membrane-spanning region of the TM.
- an isolated peptide comprising a fragment of a viral envelope protein, wherein said peptide is free of the portion of the envelope protein N- terminal of the membrane-spanning region of the envelope protein is provided by the present invention, said peptide having a membrane- destabilizing activity.
- the present invention also is directed to viral vectors or viral particles (virions) wherein the envelope protein of the virus is modified to include one or more peptides, which peptide (s) have the propensity to form amphiphilic structures, particularly amphiphilic alpha-helical structures, and may be derived from a viral envelope protein or which may be obtained from other sources, and wherein the peptide (s) is incorporated into a portion of the envelope protein that is exterior to the viral membrane.
- modified envelope proteins also may include a targeting polypeptide containing a binding region that binds to a ligand or the targeting polypeptide may be attached separately to the viral membrane.
- the peptide of the present invention aids in host or target cell entry by providing an additional membrane-active component for fusing the viral vectors or vector particles to such cells.
- the peptides of the present invention may be attached to the viral membrane of the viral vector or viral particle and such vector or viral particle may or may not include an envelope protein.
- the viral vector or viral particle includes an envelope protein
- the peptide is attached separately to the viral membrane and is not incorporated into the envelope protein.
- the envelope protein may be a wild type viral envelope protein, or may be a modified viral envelope protein including a targeting polypeptide.
- the peptide (s) of the present invention form an "artificial envelope protein.”
- the "artificial envelope protein" also includes a targeting polypeptide.
- the present invention also is directed to packaging cells and producer cells that include polynucleotides encoding the peptides of the present invention.
- packaging cells and producer cells generate modified viral vectors or viral particles as herei- nabove described that include the peptides as a portion of the viral envelope protein or in which the peptides are separately attached to the exterior and/or interior of the viral membrane.
- a viral particle to be used as a viral vector is provided with an amphiphilic peptide on the outer surface thereof and such viral par ⁇ ticle may or may not include a wild type envelope protein.
- the amphiphilic polypeptide may be incorporated into the envelope protein or may be attached to the viral membrane as an entity separate from the viral envelope protein.
- the amphiphilic peptide is attached to the viral membrane as part of an "artificial envelope protein. "
- amino acid means both natural and unnatural amino acids in either the - or D- forms. Natural amino acids are those found in nature (Morrison and Boyd, Organic Chemistry. 4 th edition, pgs. 1118-1119 (1983)). Unnatural amino acids are those not found in nature but capable of being synthesized and include, but are not limited to norleucine, norvaline, and ornithine.
- amphiphilic means that a peptide or other molecule contains both hydrophobic and hydrophilic regions.
- An amphiphilic peptide or other molecule may have a structure such that one side is hydrophobic and the other side is hydrophilic.
- the amphiphilicity of a structure within the meaning of the present invention may in particular be characterized by its hydrophbic moment ⁇ -
- polynucleotide means a polymeric form of nucleotide of any length, and includes ribonucleotides and deoxyribocleotides. This term also includes single- and double- stranded DNA, as well as single- and double-stranded RNA. In addition, the term includes modified polynucleotides , such as methylated or capped polynucleotides.
- polypeptide means a polymer of amino acids and does not refer to any particular length of polymer. Such term also includes post-translationally modified polypeptides or proteins (e.g., glycosylated, acetylated, phosphorylated, etc.).
- ligand means a molecule that is capable of being bound by a targeting polypeptide. Such molecules include, but are not limited to, cellular receptors and extracellular components such as extracellular matrix components.
- Figure 1 shows schematic representations of MoMuLV Env and its membrane-proximal region.
- A Diagram of MoMuLV Env surface subunit (SU or gp70) and transmembrane subunit (TM or pl5E) .
- the membrane- proximal region of the envelope protein i.e., amino acid residues 598-616, is shown in stripes with the corresponding amino acid sequence (shown in single letter code) .
- B The amphiphilic character of region 598-616 as predicted by the Schiffer and Edmunson's helical-wheel method (generated by DNASIS, Hitachi software) .
- C Ribbon diagram of the region 598-616 modeled as an ⁇ -helix. In the figure the hydrophilic amino acids are represented in black and the hydrophobic ones in white.
- Figure 2 provides graphs which show that peptide 598-616 of Mo- loney Murine Leukemia Virus forms an amphiphilic ⁇ -helix in the presence of membranes.
- the plot represents an average of 9 CD spectra for 50 UM peptide 598-616 in an aqueous solution (20 mM NaP0 4 ,pH 7, dashed line) , and in presence of liposomal membrane vesicles (POPG/POPC [1:3] liposomes in 20 mM Na 2 P0 4 , pH 7.4; lipid to peptide molar ratio 100:1; solid line).
- the measurements were obtained for wavelength 195-250 nm at 20 mdeg sensitivity.
- the y-axis shows the ellipticity (t ) xl0 ⁇ " (deg cm 2 dmol "
- the x-axis shows the wavelength (nm) .
- EPR electron paramagnetic resonance
- Peptide 598-616 forms a membrane-associated amphiphilic ⁇ -helix as determined by EPR.
- the parameter ⁇ was derived for the peptide 598-616 with a single residue (X-axis) substituted by Cys and spin- labeled for analysis with the nitroxide
- Figure 3 shows induction of current flux induction across a planar membrane by the wild type or native peptides 598-616, 617- 632, or a mutant peptide 598-616 R609C (final peptide concentration
- Figure 4 shows the effect of mutations in the Env cytoplasmic tail region on Env ability to induce cell-to-cell fusion.
- the indicator XC6 cells were added 24 hours post-transfection, were fixed 12 hours later and scored microscopically on ten 2 mm 2 grids for syncytia (cells with four or more nuclei).
- the left y-axis indicates the titers.
- the right y- axis indicates the fusion in % cell to cell fusion.
- NIH3T3 cells were photographed 24 hours post-transfection with the R-less Env constructs containing wild type membrane-proximal region 598-616 (picture no. I), or substituted with the melittin fragment (picture no. II), the hydrophilic (picture no. Ill), or the random sequences (picture no . IV) .
- Figure 5 shows the efficiency of particle incorporation for Env mutants with truncations (A) , or point mutations (B) , or substitutions (C) in cytoplasmic tail region.
- Supernatant from 293T culture transiently transfected with the three expression plasimids encoding the env, gag-pol, and ⁇ -gal genes was the source of virions used for Western Blot analysis with anti- gp 70, anti- p 30, and/or anti- pl5 E antibodies.
- the mutant Env used in transfections are indicated above the gels. H 2 0-mock transfected.
- panel (A) the top half of the gel was exposed only to the anti -gp70 antibody and the bottom to the anti- gp30 and anti-pl5E antibodies.
- Figure 6 shows a hypothetical model of the MoMuLV envelope protein sub-ectodomain region (i.e. , the memberane-spinning region and the cytoplasimic tail region) .
- A The monomer of the submembrane (i.e. , the cytoplasimic tail region) envelope protein segment before and after the R peptide cleavage as described herein. The position of Arg 609 is shaded, hydrophobic regions are in white.
- B The proposed sub-ectodomain unit shown as a trimer of two unprocessed tails and one R-less tail.
- C HIV-1 matrix trimer as crystallized by Hill, et al . , 1995. The representation is based on the coordinates obtained from the Brookhaven web site, Accession No. 1 HIW.
- an isolated peptide in one embodiment, com- prises an amino acid sequence derived or isolated from a viral envelope protein, wherein such peptide forms an amphiphilic structure.
- the peptide is derived from a viral envelope protein amino acid sequence, at least a portion of which is located in the cytoplasmic tail region and adjacent to the membrane-spanning region of the transmembrane subunit of the envelope protein.
- such peptide comprises a fragment of a viral envelope protein which is free of the SU and the external region of the TM, or all sequences 5' of the membrane spanning region of the transmembrane subunit.
- this fragment includes at least the first four amino acids of the N-terminal portion of the cytoplasmic tail region of the transmembrane subunit of the envelope protein.
- Preferred is an embodiment which includes at least the first six amino acids of the N-terminal portion of the cytoplasmic tail region of the transmembrane subunit of the envelope protein. More preferred is an embodiment including the first eight amino acids.
- the peptide comprises an amino acid sequence that is a derivative or analogue of the amino acid sequence hereinabove described.
- the derivative or analogue may have at least one substitution of an amino acid residue of the above- mentioned amino acid sequence.
- the analogue may include a reverse amino acid sequence as compared to the amino acid sequence present in the env protein from which it is derived.
- the derivative or analogue may either include D or L amino acids.
- analogues of the peptides of the invention that employ other backbones than pep- tidic backbones and retain the overall stereochemical positions of the R-groups of the peptides.
- An example of such a backbone is a peptide-amide backbone.
- Polynucleotides may also provide such a backbone, e.g. a methylphosphonate backbone may serve as the backbone structure carrying the relevant R-groups.
- the peptide is comprised of an amino acid sequence derived from the cytoplasmic tail region of the TM as hereinabove described, and further includes at least a portion of the membrane-spanning region of the TM.
- the isolated peptides of the present invention are amphiphilic peptides, which may have an alpha-helical secondary structure, especially in the presence of membranes. Alternatively, the isolated amphiphilic peptides may have a different secondary structure, such as a beta sheet .
- the peptides, derivatives and analogues of peptides contemplated in the present invention have membrane destabilizing activity.
- Various methods of determining the membrane destabilizing activity of a compound are known to the person skilled in the art and may be employed to determine the membrane destabilizing activity within the meaning of the present invention.
- elec- trophysiological methods of determining the membrane destabilizing activity may be used. Such methods include measuring the release of a suitable marker, such as e.g. a cation, such as e.g. potassium, from a liposome under defined conditions (see e.g. example 1) . Release of a suitable fluorescent marker from a liposome may also be measured in a suitable assay.
- An alternative assay is the planar lipid bilayer integrity assay as known in the art and described in example 1.
- peptides wherein the membrane-destabilizing activity of said peptide is sufficient to induce an detectable increase of the release of a suitable marker from a liposome are part of this invention.
- a detectable increase will occur at an active concentration of 30 mM peptide / 1 mol lipid in a suitable electrophysiological assay.
- Preferred is a peptide that will show such an increase at an active concentration of 10 mM peptide / 1 mol lipid.
- Most preferred is a peptide that will show such an increase at an active concentration of 0.5 mM peptide / 1 mol lipid.
- the potassium release from a liposome as measured in a potassium release assay as essentially described in example 1 will be more than 10%. In a more preferred embodiment it will be more than 20% under the same conditions.
- the preferred peptides of the invention have an amphiphilic structure, in particular an ⁇ -helical amphiphilic structure.
- the peptides, derivatives and analogues of peptides contemplated in the present invention have an amphiphilic structure with a hydrophobic moment of at least 0.9 as calculated using the DNASIS software employing the Chou, Fasman and Rose algorithm and calculated with the Kyte and Doolittle algorithm.
- Preferred is a hydrophobic moment of at least 1.0.
- the present invention also contemplates the use of an amphiphilic compound other than a peptide, derivative or analogue of a peptide having a membrane-destabilizing activity for the preparation of a viral vector.
- compounds wherein the membrane-destabilizing activity of said compound is sufficient to induce a detectable increase of the release of a suitable marker from a liposome are part of this invention.
- a detectable increase will occur at an active concentration of 30 mM compound / 1 mol lipid in a suitable electrophysiological assay.
- Preferred is a compound that will show such an increase at an active concentration of 10 mM compound / 1 mol lipid.
- Most preferred is a compound that will show such an increase at an active concentration of 0.5 mM compound / 1 mol lipid.
- the preferred compounds of the invention have an amphiphilic structure.
- the compounds contemplated in the present invention have an amphiphilic structure with a hydro- phobic moment of at least 0.9 as calculated using the DNASIS software employing the Chou, Fasman and Rose algorithm and calculated with the Kyte and Doolittle algorithm.
- Preferred is a hydrophobic moment of at least 1.0.
- the isolated peptides of the present invention can be of various lengths.
- the peptides include an amphiphilic amino acid sequence having from 8 to 40 amino acid residues, in a preferred embodiment the peptides of the invention include 12 to 35 amino acid residues. In a particular preferred embodiment the peptides include at least 8 amino acid residues.
- Such isolated peptides which include amino acid residues that are derived from the cytoplasmic tail region of the TM, and may also include amino acid residue (s) from the membrane-spanning region of the TM, also sometimes are hereinafter referred to as "membrane- proximal" amphiphilic peptides. Representative examples of such peptides are given in Table I below.
- negative numbers refer to the number of amino acid residues in the C-terminal portion of the predicted membrane-spanning region of the transmembrane subunit of the viral envelope protein
- positive numbers refer to the number of amino acid residues from the cytoplasmic tail region of the TM, beginning at the N-terminal residue of the tail, that are in the peptide.
- the boundary between the membrane-spanning region of the transmembrane subunit and the cytoplasmic tail region of the TM is at the first hydrophilic amino acid residue after the stretch of about 20 hydrophobic amino acids C-terminal to the external region of the transmembrane subunit.
- a peptide denoted as "-2/14” means that the isolated peptide includes (in an N-terminal to C-terminal direction) , as the first two amino acid residues of the N- terminus, the two C- terminal amino acids of the predicted membrane-spanning region of the transmembrane subunit and, as the last 14 amino acid residues, the 14 N-terminal amino acids of the predicted cytoplasmic tail region.
- Peptides denoted by two positive numbers are peptides that are contained only in the cytoplasmic tail region of the viral envelope protein.
- a peptide denoted as "1/17” means that the isolated peptide includes the 17 N-terminal amino acids of the predicted cytoplasmic tail region.
- Table I lists the positions of the most amphiphilic membrane-proximal segments in a number of viral envelope proteins . The scope of the present invention, however, is not intended to be limited thereby. Table I also does not imply the most active size or residue identity of the segments listed therein. Also, in the membrane-proximal domains of surface viral proteins other than viral envelope proteins, analogous amphiphilic regions are detected, such as, for example, in the M2 protein of influenza virus, and in the spike protein of adenovi- rus . Non-viral lytic peptides also contain stretches of similar characteristics (e.g., melittin).
- An artificial or synthetic amphiphilic sequence can be generated to mimic the amphiphilic membrane-destabilizing properties of the wild-type peptides identified herein as illustrated by the use of a melittin analogue described below.
- the following abbreviations are used in Table I: ALV-avian leukosis virus; BLV-bovine leukemia virus; EIA-equine infectious anemia; FIV-feline immunodeficiency virus; HEP C-hepatitis C; HIV- human immunodeficiency virus; HTLV-human T-cell leukemia virus; hRSV-human respiratory syncytial virus; infM2-influenza M2virus; INF-influenza; MMTV-Mouse Mammary Tumor Virus; MPMV-Mason Pfizer monkey virus; RSV-Rous Sarcoma Virus; PINF-parainfluenza; SNV- spleen necrosis virus; VSV-vesicular stomatitis
- such peptides include at least one hydrophilic amino acid residue which is "out-of-phase" (i.e., a hydrophilic amino acid residue in a hydrophobic region of the predicted amphiphilic structure) .
- out-of-phase i.e., a hydrophilic amino acid residue in a hydrophobic region of the predicted amphiphilic structure
- Applicants do not intend to be limited by any theoretical reasoning, it is believed that, when such pep- tides are contained in the cytoplasmic tail region of a viral envelope protein, they enter a cell membrane at an oblique angle.
- a structural distortion resulting from an out-of-phase amino acid residue may be involved in providing an oblique angle needed for membrane destabilization during fusion (Martin, et al . , J. Virol. Vol. 70, pgs. 298-304).
- the resulting changes in membrane curvature thus may decrease the energy required for fusion of lipid bilayers. This mechanism may be employed and thus preserved evolutionari
- the peptide has the amino acid sequence (SEQ ID NO:l), which is as follows:
- ILNRLVQFVKDRISWQAL This peptide corresponds to amino acid residues 598-616 of the wild type envelope protein of Moloney Murine Leukemia Virus.
- the residues ILN are from the predicted membrane-spanning region of the transmembrane subunit, whereas the residues RLVQFVKDRISWQAL are from the predicted cytoplasmic tail region of the TM.
- the peptide is an analogue or derivative of (SEQ ID NO: 1) which has at least one substitution of (SEQ ID NO: 1) that maintains the amphiphilic structure and membrane destabilization activity of the peptide.
- Such peptides may be employed in providing viral vectors that include the peptides as part of a modified envelope protein, or wherein the peptides are attached separately to the exterior and/or interior of the viral membrane.
- the viral vector may or may not include a viral envelope protein.
- the peptides are part of an "artificial envelope protein. "
- the peptide that is included in the modified viral envelope protein or is attached to a viral membrane as hereinabove described is a synthetic peptide or a naturally occurring peptide which is obtained from an organism other than a virus, which peptide is a biologically active amphiphilic peptide, such as, for example, melittin peptide, magainin peptides, XPF peptides, PGLa peptides, CPF peptide, and defensins.
- the peptide is an analogue, fragment, or derivative of melittin peptide.
- such peptide has the following structural formula: LKVLTTGLPAL (X) S (W) m ( I ) n , wherein X is isoleucine or methionine, m is 0 or 1, and n is 0 or 1.
- X is methionine
- each of m and n is 0, and the peptide has the following structure: LKVLTTGLPALMS . (SEQ ID NO: 2) .
- m is l
- n is i
- X methionine
- the peptide has the following structure:
- the peptide is an analogue or derivative of (SEQ ID NO: 2) or (SEQ ID NO: 3) which may have at least one substitution that maintains the amphiphilic or alpha- helical structure and the general functional properties of the peptide.
- the analogue may include a reverse amino acid sequence as compared to the amino acid sequence present in the env protein from which it is derived.
- the derivative or analogue may either include D or L amino acids.
- amphiphilic peptides that preferably form an alpha-helical structure are used for producing an artificial envelope protein or for modifying an existing envelope protein of a viral vector.
- an enveloped virus wherein the viral envelope is modified to include the amphiphilic peptide hereinabove described at one or more locations of the exterior portion of the viral envelope.
- the amphiphilic peptide aids in fusing the virus to cells.
- the modified viral envelope further includes a targeting polypeptide containing a binding region that binds to a ligand.
- Enveloped viruses that may include the amphiphilic peptide, and a targeting polypeptide, if desired, in the viral envelope include, but are not limited to, enveloped RNA viruses and enveloped DNA viruses.
- Enveloped RNA viruses include, but are not limited to, retroviruses (including murine leukemia viruses and gibbon ape leukemia virus); alphaviruses (including Sindbis virus); arenaviruses ; orthomyxoviruses; paramyxoviruses ; and coronaviruses .
- Enveloped DNA viruses include, but are not limited to, Herpes viruses (including Herpes Simplex Virus) and poxviruses .
- the isolated peptides of the present inventon are derived from the cytoplasmic tail region of the viral envelope protein and may or may not include amino acids derived from the membrane-spanning region of the viral envelope protein.
- the enveloped virus is a retrovirus.
- the amphiphilic peptide is incorporated into the envelope protein in a region that is neither the cytoplasmic tail region nor the membrane-spanning region of the transmembrane subunit.
- the amphiphilic peptide may be located in any position in the envelope protein that is suitable for presenting the peptide in a functional manner.
- the peptide is placed at the N-terminal end of the surface subunit of the envelope protein.
- the envelope protein is a Moloney Murine Leukemia Virus envelope protein
- the peptide may be placed between amino acid residues 6 and 7 of the receptor binding region or at the N-terminus Bstl site located between residues 16 and 17 of the receptor binding region.
- the peptide also may be inserted into or substituted for conserved exposed cysteine-constrained loops of the envelope protein (e.g. in the region of residues 74-84, or 177-181) of the receptor binding region.
- the exposed loops as recently identified based on the crystallographic resolution of the tropism-determining segment from the Friend Murine Leukemia Virus (Fass, et al . , Science Vol. 277, Pgs.1662-1666, (1997) may be useful for the insertion of the functional peptides into the envelope protein.
- the peptide also may be inserted into the hypervariable polyproline or "hinge" region of the envelope protein.
- amino acid residues 34 through 49 of the hypervariable polyproline region of the Moloney Murine Leukemia Virus envelope protein are removed and replaced with a peptide as hereinabove described.
- the peptide is inserted between amino acid residues 35 and 36 of the hypervariable polyproline region the Moloney Murine Leukemia Virus envelope protein.
- amphiphilic peptide may precede the first N-terminal residue of the SU. In yet a further embodiment, the amphiphilic peptide may be after the last C-terminal residue of the SU.
- a polynucleotide encoding a modified envelope protein which includes the amphiphilic peptide hereinabove described, wherein the amphiphilic peptide, in addition to being present in the cytoplasmic tail region of the TM, also is present in an external portion of the envelope protein at one or more positions.
- the modified envelope protein also may include a targeting polypeptide, as hereinabove described.
- Such a polynucleotide may be constructed in accordance with genetic engineering techniques known to those skilled in the art.
- the polynucleotide encoding the modified envelope protein includes the nucleic acid sequence (SEQ ID NO: 4), or a degenerate sequence thereof.
- the polynucleotide encoding the modified envelope protein includes the nucleic acid sequence (SEQ ID NO: 5) , or a degenerate sequence thereof.
- the polynucleotide encoding the modified envelope protein includes the nucleic acid sequence (SEQ ID NO: 6) , or a degenerate sequence thereof.
- Such a polynucleotide as hereinabove described may be employed in the generation of the viral vectors or viral particles described hereinabove.
- Such viral vectors or viral particles of the present invention may be constructed by a variety of methods known to those skilled in the art.
- viral vectors or viral particles may be generated from packaging cells and producer cells that include polynucleotides encoding the retroviral gag and pol proteins, and one or more polynucleotides that encode the components of the modified viral envelope proteins hereinabove described.
- the polynucleotide encoding the modified envelope protein, which includes the amphiphilic peptide may be contained in an appropriate expression vehicle, such as a retroviral expression plasmid, such as those further described herein, which is transfected into an appropriate "pre-packaging" cell line that includes nucleic acid sequences encoding the retroviral gag and pol proteins, whereby the "pre-packaging" cell line becomes a packaging cell line.
- "pre-packaging" cell lines that may be transfected with the polynucleotide encoding the modified envelope pro- tein, include GP8 cells, GPL cells, and GPNZ cells as described in Morgan, et al . , J. Virol.. Vol. 67, No. 8, pgs. 4712-4721 (August 1993) .
- the polynucleotide may be transfected into the pre-packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
- the resulting packaging cells may be transfected with an appropriate retroviral expression plasmid, such as those described herein, and that may include a polynucleotide encoding a therapeutic agent by means known to those skilled in the art, to form a producer cell line.
- Such producer cells generate infectious retroviral vector particles that include the modified envelope protein hereinabove described, in which the amphiphilic peptide is located in the external portion of the viral envelope protein as well as in the cytoplasmic tail region.
- a polynucleotide encoding a modified envelope protein that includes the amphiphilic peptide and the targeting polypeptide is contained in an appropriate expression vehicle, and is transfected into an appropriate pre-packaging cell line as hereinabove described to form a packaging cell.
- the packaging cell then may be transfected with an appropriate expression vehicle such as those described herein to form a producer cell, which generates infectious retroviral particles that include a modified envelope protein that includes the amphiphilic peptide and the targeting polypeptide in the external portion of the retroviral envelope protein.
- a retroviral particle that includes a retroviral envelope protein.
- the retroviral envelope protein may be an unmodified wild type retroviral envelope protein, or may be a modified retroviral envelope protein that includes a targeting polypeptide, wherein a portion of the viral envelope protein is replaced with a targeting polypeptide.
- the retroviral particle also includes the amphiphilic peptide, which is attached separately to the viral membrane.
- the amphiphilic peptide is attached separately to the viral membrane via an anchor comprised of at least a portion of a membrane- spanning region of a viral envelope protein such as, for example, the membrane-spanning region of the TM of a retroviral envelope protein.
- the amphiphilic peptide is at- tached separately to the viral membrane by chemical means, such as those described below.
- such a retroviral particle may be generated by transfecting a pre-packaging cell line with a first polynucleotide and a second polynucleotide.
- the first polynucleotide encodes an unmodified wild type retroviral envelope protein or a modified viral envelope protein that includes a targeting polypeptide as hereinabove described.
- Such polynucleotide may be contained in a retroviral expression plasmid.
- the second polynucleotide includes a nucleic acid sequence encoding the amphiphilic peptide hereinabove described, and a nucleic acid sequence encoding at least a portion, and in one embodiment, all, of the membrane-spanning region of the transmembrane subunit of a viral envelope protein with or without nucleic acid sequences encoding the cytoplasmic tail region of the TM.
- the nucleic acid sequence encoding the membrane-spanning region of the transmembrane subunit is located 5' to the nucleic acid sequence encoding the amphiphilic peptide.
- the nucleic acid sequence encoding the membrane- spanning region of the transmembrane subunit is located 3 ' to the nucleic acid sequence encoding the amphiphilic peptide.
- a packaging cell line Upon transfection of a pre-packaging cell with the first and second polynucleotides, a packaging cell line is formed.
- a producer cell line then may be formed from the packaging cell line by means known to those skilled in the art.
- the resulting producer cell line generates viral particles that include the modified envelope protein including the targeting polypeptide.
- the amphiphilic peptide is attached to the viral membrane as an entity separate from the viral envelope protein, on either the exterior or the interior of the viral membrane or on both sides of the viral membrane.
- a viral vector or viral particle including a modified envelope protein, including a targeting polypeptide may be generated from a pre-packaging cell as hereinabove described.
- the amphiphilic peptide then is attached to the viral membrane by chemical means .
- the amphiphilic peptide of the present invention may be attached to the viral membrane first by forming a peptide-lipid conjugate.
- a conjugate may be formed by ligating a lipid such as, for example, a lipid having a maleimi- doyl moiety, to an amino group in the peptide.
- the conjugate may be prepared according to the standard protocols in an aprotic sol- vent . After the reaction is completed, preliminary purification may be achieved by gel filtration on Sephadex LH-20 in dimethyl- formamide followed by precipitation of the conjugate with ether. The purity of the conjugate then is verified by mass spectrometry .
- the attachment of the conjugate to the viral membrane is carried out by mixing small quantities of the conjugate, dissolved in acetonitrile, with the viral particles.
- the amount of conjugate should not exceed 10 to 15% of the total amount of lipid in the resulting modified viral envelope.
- the viral particles now including the amphiphilic peptide attached to the viral membrane may be purified by means known to those skilled in the art.
- a lipid-polyethylene glycol (PEG) - amphiphilic peptide conjugate may be attached to the viral membrane.
- a lipid-peptide conjugate such as hereinabove described may be attached to a polyethylene glycol polymer having a molecular weight of about 2,000 and bearing a distal sulfhydryl group, to form a lipid - PEG - peptide conjugate.
- the conjugate then can be purified by employing gel-filtration chromatrography in an aprotic medium (e.g., Sephadex LH-20 in DMF) , or by employing gel filtration/absorption chromatography on a Toyopearl HW-40 (Toyo Soda, Japan) in DMF, tetrahydrofuran, or methanol.
- an aprotic medium e.g., Sephadex LH-20 in DMF
- Toyopearl HW-40 Toyo Soda, Japan
- the lipid - PEG - peptide conjugate may be attached to the viral membrane by mixing a small quantity of a solution of the lipid - PEG - peptide conjugate in acetonitrile with the viral particles.
- the resulting viral particles thus have the amphiphilic peptide attached to the viral membrane. Because the resulting viral particles also include polyethylene glycol, the resulting viral particles also will be less likely to be recognized by the immune system.
- a retroviral vector particle which includes a naturally occurring or wild-type or native, retroviral envelope protein.
- retroviral vector particle also includes the amphiphilic peptide and the targeting polypeptide hereinabove described, wherein the amphiphilic peptide and the targeting polypeptide are attached to the viral membrane.
- each of the amphiphilic peptide and the targeting polypeptide is attached individually to the viral membrane. Such attachment may be through an anchor comprised of at least a portion of the membrane-spanning region of a transmembrane subunit of a viral envelope protein, or through a glycolipid linker, or through a peptide - lipid conjugate as hereinabove described.
- a polypeptide is formed which includes the targeting polypeptide, the amphiphilic peptide, and a spacer moiety, such as, for example, a Glycine-Serine-Glycine tripeptide placed between the targeting polypeptide and the amphiphilic peptide.
- the resulting polypeptide is attached to the viral membrane. Such attachment may be accomplished via at least a portion of the membrane-spanning re ⁇ gion of a transmembrane subunit, a glycolipid linker, or through a peptide-lipid conjugate as hereinabove described.
- such a retroviral vector particle may be constructed by transfect- ing a packaging cell line such as those hereinabove described which includes polynucleotides encoding gag, pol, and env proteins, with expression plasmids including a first polynucleotide and a second polynucleotide.
- packaging cell lines include, but are not limited to, the PE501, PA317 (ATCC No. CRL 9078), ⁇ -AM, PA12 ,
- the first polynucleotide includes a nucleic acid sequence encoding the amphiphilic peptide hereinabove described and a nucleic acid sequence encoding at least a portion and, in one embodiment, all, of the membrane-spanning region of a transmembrane subunit of a viral envelope protein with or without nucleic acid sequences encoding the cytoplasmic tail.
- the second polynucleotide includes a nucleic acid sequence encoding a targeting polypeptide as hereinabove described and a nucleic acid sequence encoding at least a portion and, in one embodiment, all, of the membrane-spanning region of a transmembrane subunit of a viral envelope protein with or without nucleic acid sequences encoding a portion or all of the cytoplasmic tail region of the TM.
- a producer cell line then may be formed by means known to those skilled in the art.
- the resulting producer cells generate infectious retroviral vector particles that include the wild-type retroviral envelope protein and which exhibit altered receptor specificity and greater fusogenicity via the individually attached targeting peptide and amphiphilic fusion peptide, respec ⁇ tively.
- such viral particles including the attached targeting polypeptide may be generated by transfecting a prepackaging cell line with the first and second polynucleotides and a polynucleotide encoding wild-type envelope protein.
- the packaging cell line may be transfected with a single polynucleotide including a nucleic acid sequence encoding at least a portion and, in one embodiment, all, of a membrane-spanning region of a transmembrane subunit, a nucleic acid sequence encoding the amphiphilic peptide, a nucleic acid sequence encoding a spacer moiety, and a nucleic acid sequence encoding the targeting polypeptide.
- a producer cell then is formed by means known to those skilled in the art.
- the resulting producer cell generates viral particles that include a wild-type envelope protein, wherein the polypeptide including the amphiphilic peptide and the targeting polypeptide are attached separately to the viral membrane, whereby the amphiphilic peptide and the targeting polypeptide are exposed on the outside of the viral particle.
- the retroviral vector particle may be constructed first by generating a wild-type retrovirus from a packaging cell line such as those hereinabove described.
- the amphiphilic peptide and the targeting polypeptide each are attached to the viral membrane, either by attachment to the membrane through a peptide-lipid conjugate as hereinabove described, or through a gly- colipid linker.
- a polypeptide including the targeting polypeptide, the amphiphilic peptide, and a spacer moiety as hereinabove described is attached to the viral membrane of a wild-type retroviral particle by means such as those hereinabove described.
- a viral particle that does not include a naturally occurring or wild-type envelope protein or a modified envelope protein.
- an "artificial envelope protein" comprised of a targeting polypeptide and an amphiphilic peptide as hereinabove described.
- the targeting polypeptide and the amphiphilic peptide are attached to the viral membrane. Means of attachment include those hereinabove described.
- the targeting polypeptide and the amphiphilic peptide may be attached to the viral membrane as two independent peptides or as one polypeptide that provides both binding and fusion functions in tandem.
- the polypeptide also includes an appropriate spacer moiety placed between the targeting polypeptide and the amphiphilic peptide.
- the targeting polypeptide and the amphiphilic peptide are included as part of an "artificial envelope protein".
- one or more types of targeting and/or fusion promoting amphiphilic peptides may be included as part of the "artificial envelope protein" attached to the viral membrane for potentiating infection by the viral particle.
- Such may include the use of more than one amphiphilic peptide that promotes fusion and/or subsequent events in the infection of cells, resulting in the delivery of genetic material into the cell .
- Such a viral particle which includes an "artificial envelope protein, " may be generated by transfecting a pre-packaging cell line, including polynucleotides encoding the retroviral gag and pol proteins as hereinabove described, with a first polynucleotide including a nucleic acid sequence encoding the amphiphilic peptide and a nucleic acid sequence encoding a portion or all of the membrane-spanning region of the transmembrane subunit of a viral envelope protein with or without a nucleic acid sequence encoding the cytoplasmic tail region, and a second polynucleotide including a nucleic acid sequence encoding a targeting polypeptide and a nucleic acid sequence encoding a portion or all of the membrane- spanning region of the transmembrane subunit of a viral envelope protein with or without a nucleic acid sequence encoding the cytoplasmic tail region.
- the pre-packaging cell line is transfected with a single polynucleotide including a nucleic acid sequence encoding a portion or all of the membrane-spanning region of the transmembrane subunit, a nucleic acid sequence encoding the amphiphilic peptide, a nucleic acid sequence encoding a spacer moiety, and a nucleic acid sequence encoding the targeting polypeptide.
- a producer cell line then may be formed by transfecting the pre-packaging cell line with an appropriate retroviral expression plasmid, such as those herein described.
- the producer cell Upon transfection of the pre-packaging cell with the appropriate polynucleotide (s) , and an appropriate retroviral expression plasmid as described herein to form a producer cell, the producer cell generates viral particles which include an "artificial envelope protein, " including the amphiphilic peptide and the targeting polypeptide, attached to the viral membrane.
- an "artificial envelope protein, " including the amphiphilic peptide and the targeting polypeptide is attached to the viral membrane separately.
- a single polypeptide including the amphiphilic peptide and the targeting polypeptide is attached to the viral membrane.
- a viral particle is generated from a prepackaging cell line.
- Such viral particle includes a viral membrane, but does not include a viral envelope protein.
- each of the amphiphilic peptide and the targeting polypeptide is attached to the viral membrane by means such as those hereinabove described, such as, for example, by attaching the amphiphilic peptide and the targeting polypeptide to the membrane through a peptide-lipid complex, or by attaching the amphiphilic peptide and the targeting polypeptide to a viral membrane via a glycolipid linker.
- a single polypeptide including the amphiphilic peptide, a targeting polypeptide, and a spacer moiety, is attached to the viral membrane via chemical means such as those hereinabove described, to provide a viral vector particle having an "artificial envelope protein" including the amphiphilic peptide and the targeting polypeptide.
- the "artificial envelope protein” has a significantly reduced amount of material that is derived from a retroviral envelope protein, a viral particle having such an "artificial envelope protein” is less likely to elicit an immune response than a viral particle that retains all or a majority of the wild-type envelope protein structure.
- amphiphilic peptides of the present invention are employed in the formation of a variety of viral vectors or viral particles having modified viral envelopes or "artificial envelope proteins.”
- the use of such vectors employing amphiphilic peptides, derivatives or analogues of the present invention for increasing the expression of a heterologous gene transfected into a cell with the help of such a vector is contemplated by the present invention.
- Preferred vectors contemplated by the present invention are such vectors that increase the expression of a heterologous gene by more than lOfold, as compared to a suitable control, such as a corresponding vector that does not employ an amphiphilic peptide, derivative or analogue of the present invention.
- the "artificial envelope" of the viral particle can be generated via expression of the targeting and fusion peptides on the surface of the viral particle as hereinabove described.
- an artificial surface may be generated, for example, as an artificial bilayer used to envelop viral particles derived by any means. This constitutes the generation of artificial virusomes that can be retargeted and/or engineered to have enhanced fusion or other entry parameters due to the new encapsulating surface.
- the amphiphilic peptides described herein or analogues thereof may serve a variety of functions.
- the peptide may function as a fusion potentiating molecule.
- the peptides provide for more efficient incorporation of external polypeptides into a viral surface coat.
- the targeting polypeptide which may be included in the various embodiments of the vector particles hereinabove described, includes a binding region that binds to a receptor located on a desired cell type.
- Such targeting polypeptides include, but are not limited to, antibodies and fragments thereof, including single-chain antibodies, monoclonal antibodies, and polyclonal antibodies.
- Such antibodies include, but are not limited to, antibodies and fragments or portions thereof which bind to erb-B2 , such as, for example, e23 antibody; antibodies which bind to receptors such as, for example, the CD4 receptor on T-cells; antibodies which bind to the transferring receptor; antibodies directed against human leukocyte antigen (HLA) ; antibodies to carcinoembryonic antigen; antibodies to pla- cental alkaline phosphates found on testicular and ovarian cancer cells; antibodies to high molecular weight melanoma-associated antigen; antibodies to polymorphic epithelial mucin found on ovarian cancer cells; antibodies to human chronic gonadotropin; antibodies to CD20 antigen of B-lymphoma cells; antibodies to alpha- fetoprotein; antibodies to prostate specific antigen; OKT-3 antibody, which binds to CD3 T-lymphocyte surface antigen; antibodies which bind to B-lymphocyte surface antigen; antibodies which bind to EGFR (c-erb-Bl
- cytokines include, but are not limited to, interleukins, including Interleukin-l ⁇ , Interleukin-l ⁇ , and Interleukins 2 through 14; growth factors such as epithelial growth factor (EGF) , TGF- ⁇ , TGF- ⁇ , fibroblast growth factor (FGF) , keratinocyte growth factor (KGF) , PDGF-A, PDGF-B, PD-ECGF, IGF-I, IGF-II, and nerve growth factor (NGF) , which binds to the NGF receptor of neural cells; colony stimulating factors such as GM-CSF, G-CSF, and M-CSF, leukemia inhibitory factor (LIF) ; interferon's such as interferon- ⁇ , interferon- ⁇ , and interferon- ⁇ ; inhibin A; inhibin B; chemotac- tic factors; ⁇ -type intercrine cytokines
- Still other targeting polypeptides which may be employed include, but are not limited to, melanoma stimulating hormone (MSH) , which binds to the MSH receptor on melanoma cells; peptidomimetic analogues of Ot-MSH, including a peptidomimetic analogue having the structure Ser-Tyr-Ser-Nle-Glu-His- (D-Phe) -Arg-Trp-Gly-Lys-Pro-Val , wherein Nle is norleucine and D-Phe is a D-phenylalanine residue; the polypeptide FLA16, which has the sequence Cys-Gln-Ala-Gly-Thr- Phe- A la-Leu-Arg-Gly-Asp-Asn-Pro-Gln-Gly-Cys, which binds to the in- tegrins VLA3 , VLA4 , and VLA5 found on human histiocytic lymphoma cells
- the targeting polypeptide is a single chain antibody.
- the targeting polypeptide includes a binding region that binds to an extracellular matrix component.
- extracellular matrix component means a molecule that occupies the extracellular spaces of tissues.
- extracellular matrix components include, but are not limited to, collagen (including collagen Type I and collagen Type IV) , laminin, fibronectin, elastin, glycosaminoglycans , proteoglycans, and sequences which bind to fibronectin, such as arginine-glycine- aspartic acid, or RGD, sequences.
- Binding regions that bind to an extracellular matrix component, and which may be included in a targeting polypeptide include, but are not limited to, polypeptide domains that are functional domains within von Willebrand Factor or derivatives thereof, wherein such polypeptide domains bind to collagen.
- the binding region is a polypeptide having the following structural formula: Trp-Arg-Glu-Pro-Ser-Phe-Met- Ala-Leu-Ser .
- binding regions that bind to an extracellular matrix component include, but are not limited to, the arginine-glycine-aspartic acid, or RGD, sequences, which binds fibronectin, and a polypeptide having the sequence Gly-Gly-Trp-Ser-His-Trp, which also binds to fibronectin.
- the viral vector or viral particle further includes at least one polynucleotide encoding a heterologous polypeptide that is to be expressed in a desired cell.
- the heterologous polypeptide may, in one embodiment, be a therapeutic agent.
- therapeutic is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents .
- the polynucleotide encoding the therapeutic agent is under the control of a suitable promoter.
- suitable promoters that may be employed include those known to those skilled in the art, including, but are not limited to, the retroviral LTR; the SV40 promoter; the cytomegalovirus (CMV) promoter; and the Rous Sarcoma Virus (RSV) promoter.
- the promoter also may be the native promoter that controls the polynucleotide encoding the therapeutic agent. It is to be understood, however, that the scope of the present invention is not to be limited to specific foreign genes or promoters.
- the polynucleotide encoding a therapeutic agent may be contained in a retroviral expression plasmid, which is transfected into the appropriate packaging or pre ⁇ packaging cells hereinabove described, to form producer cells that generate the vector particles hereinabove described.
- the retroviral expression plasmid may be derived from Moloney Murine Leukemia Virus and is of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al . , J. Virol., Vol. 61, pgs. 1639-1649 (1987) and Miller, et al . , Biotechniques , Vol. 7, pgs 980-990 (1989).
- the retroviral expression plasmid may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs.
- the restriction product has an average DNA size of at least 10,000 base pairs.
- the retroviral expression plasmid includes one or more promoters for the genes contained in the vector. 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, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters) .
- CMV human cytomegalovirus
- viral promoters that may be employed include, but are not limited to, adeno- virus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
- the viral vectors or viral particles which include the amphiphilic peptide hereinabove described and may further include a targeting polypeptide, and a polynucleotide encoding a therapeutic agent, may be administered to a host in an amount effective to produce a therapeutic or beneficial effect in the host.
- beneficial effect means that the effect is less than curative, but improves the quality of life in the host, such as, for example, alleviating a medical condition.
- the host may be a mammalian host, which may be a human or non-human primate host.
- the viral vectors or viral particles upon administration to the host, travel to and transduce the desired cells, whereby the transduced target cells express the therapeutic agent in vivo .
- the exact dosage of viral vectors or viral particles that may be administered is dependent upon a variety of factors, including the age, sex, and weight of the patient, the target cells which are to be transduced, the therapeutic agent that is to be administered, and the severity of the disease or disorder to be treated.
- compositions suitable for medical treatment that include a peptide of the invention or a viral or synthetic vector of the invention, are also within the scope of the present invention.
- the viral vectors or viral particles or compositions including such viral vectors or viral particles may be administered to the host systemically, such as, for example, by intravenous, intraperi- toneal, intracolonic, intratracheal , endotracheal, intranasal, in- travascular, intrathecal, intraarterial, intracranial, intramarrow, intravesicular, intrapleural, intradermal, subcutaneous, intramuscular, intraocular, intraosseous, and intrasynovial administration.
- the viral vectors or viral particles also may be administrated topically.
- Cells that may be transduced with the viral vectors or viral particles of the present invention include, but are not limited to, primary cells, such as primary nucleated blood cells, primary tumor cells, endothelial cells, epithelial cells, vascular cells, kerati- nocytes, stem cells, hepatocytes, chondrocytes , connective tissue cells, fibroblasts and fibroelastic cells of connective tissues, mesenchymal cells, mesothelial cells, and parenchymal cells; smooth muscle cells of the vasculature; hematopoietic stem cells; T- lymphocytes; B-lymphocytes; neutrophils; macrophages; platelets; erythrocytes; reparative mononuclear granulocytic infiltrates of inflamed tissues; nerve cells; brain cells; muscle cells; osteo- cytes and osteoblasts in bone; lung cells, pancreatic cells; epithelial and subepithelial cells of the gastrointestinal and
- the selection of the particular cells which are to be transduced is dependent upon the disease or disorder to be treated as well as the targeting polypeptide. Such cells may be transduced in vivo, or may be transduced ex vivo, and then administered to a host in an amount effective to provide a therapeutic effect or a beneficial effect. It is to be understood that the scope of the present invention is not to be limited to the transduction of any specific cells .
- viral vectors or viral particles include a targeting polypeptide that binds to an extracellular matrix component
- viral vectors or viral particles may be employed in treating diseases or disorders associated with an exposed extracellular matrix component.
- diseases or disorders include, but are not limited to, cardiovascular diseases; cirrhosis of the liver; connective tissue disorders (including those associated with ligaments, tendons, and cartilage) ; and vascular disorders associated with the exposition of collagen.
- the vector particles may be used to deliver therapeutic genes to restore endothelial cell function and to combat thrombosis, in addition to limiting the proliferative and fibrotic responses associated with neointima formation.
- the vector particles also may be employed in treating vascular lesions; ul- cerative lesions; areas of inflammation; sites of laser injury, such as the eye, for example; sites of surgery; arthritic joints; scars; and keloids.
- the viral vectors or viral particles also may be employed in wound healing.
- viral vectors or viral particles which include a targeting polypeptide that binds to an extracellular matrix component also may be employed in the treatment of tumors, including malignant and non-malignant tumors.
- tumors when invading normal tissues or organs, secrete enzymes such as colla- genases or metalloproteinases that expose extracellular matrix components.
- enzymes such as colla- genases or metalloproteinases that expose extracellular matrix components.
- Such tumors include, but are not limited to, carcinomas; sarcomas, including chondrosarcoma, osteosarcoma, and fibrosarcoma; and brain tumors.
- a viral vector or viral particle including the amphiphilic peptide including a targeting polypeptide that binds to an extracellular matrix component located at a tumor site, and a polynucleotide encoding a negative selective marker or "suicide" gene, such as, for example, the Herpes Simplex Virus thy- midine kinase (TK) gene, may be administered to a patient, whereby the viral vector transduces the tumor cells.
- an interaction agent or prodrug such as gancyclovir or acyclovir, is administered to the patient, whereby the transduced tumor cells are killed.
- the viral vectors or viral particles which include the amphiphilic peptide, and may further include a targeting polypeptide, and a polynucleotide encoding a therapeutic agent, may be administered to an animal in vivo as part of an animal model for the study of the effectiveness of a gene therapy treatment.
- the vectors or particles may be administered in varying doses to different animals of the same species, whereby the vector particles will transduce the desired target cells in the animal.
- the animals then are evaluated for the expression of the desired therapeutic agent in vivo in the animal. From the data obtained from such evaluations, one may determine the amount of vector particles to be administered to a human patient.
- the viral vectors or viral particles of the present invention also may be employed in the in vi tro transduction of desired target cells, which are contained in a cell culture containing a mixture of cells. Upon transduction of the target cells in vi tro, the target cells produce the therapeutic agent or protein in vi tro . The therapeutic agent or protein then may be obtained from the cell culture by means known to those skilled in the art.
- the viral vectors also may be employed for the transduction of cells in vi tro in order to study the mechanism of the genetic engineering of cells in vi tro .
- amphiphilic peptide, and the targeting polypeptide if desired is incorporated into or attached to the surface of a drug delivery or nucleic acid delivery vehicle (e.g., a nanoparticle) or incorporated into or attached to the surface of an encapsulating vesicle such as a liposome.
- a drug delivery or nucleic acid delivery vehicle e.g., a nanoparticle
- an encapsulating vesicle such as a liposome.
- the peptide forms a portion of the particle or of the encapsulating vesicle.
- the peptide may be bound to the particle covalently or non-covalently, and such bonding may be achieved by physical or chemical means, including but not limited to those hereinabove described.
- the amphiphilic peptides may be associated with a liposome bilayer.
- the peptides may be attached or incorpo- rated into the inner and/or outer surfaces of the liposome bilayer by means known to those skilled in the art, such as by covalent bonding, or by linker moieties or by other means.
- the attachment of the peptides to the liposome may be to the phospholipids , lipids, lipid intricolating molecules, lipid modification molecules, or by any other means which allows surface association.
- the liposomes that include the peptides or analogues thereof may be employed for the enhanced delivery of therapeutic agents or polynucleotides to cells, or to interstitial spaces and other locations.
- the peptides or analogues thereof aid in fusing the liposome to desired cells or in releasing encapsulated therapeutic agents at a desired site.
- amphiphilic peptides may be associated with polycations or cationic polymers, such as e.g. protamine, polyethy1imine or polylysine. Polycations or cationic polymers are useful for condensing nucleic acids. Accordingly, in a further embodiment of this invention, the amphiphilic peptides may be associated with cationic lipid complexes of nucleic acids.
- Polynucleotides encoding therapeutic agents which may be contained in the liposome or the cationic lipid complex, include, but are not limited to, those described herein.
- peptides of the invention is contemplated in combination with either viral vectors or synthetic vectors, as well as with hybrid synthetic and viral vectors, such as viral vectors that are chemically modified after they have been produced by a suitable producer cell.
- amphiphilic peptides of the present invention also may be employed as antibiotics, or anti-viral agents, or antimicrobial agents, whereby such peptides reduce, inhibit, prevent, or destroy the growth of a cell, virus, or virally-infected cell.
- the peptides may be administered in vivo or in vitro.
- the peptides also may be administered directly to a target cell, virus, or infected cell, or the peptides may be administered systemically, directly or as conjugated to delivery vehicles.
- the polyvalent presentation on a surface of particles presenting the peptides is likely to potentiate the therapeutic or beneficial effect of the amphiphilic peptide or the analogues.
- the peptides of the present invention allow a method for treating or controlling microbial infection caused by organisms that are sensitive to the peptides.
- Such treatment may comprise administering to a host organism or tissue susceptible to or affiliated with a microbial infection an antimicrobial amount of at least one of the peptides .
- antibiotics because of the antibiotic, antimicrobial, and antiviral properties of the peptides, they may also be used as preservatives or sterilants of materials susceptible to microbial or viral contamination.
- the peptide (s) of the present invention may be administered to a host; in particular a human or non-human animal, in an effective antibiotic and/or anti-tumor and/or anti-viral and/or antimicrobial and/or anti-fungal and/or anti-parasitic amount.
- NIH3T3, 293T, and XC cells were obtained from the ATCC repository.
- XC6 cells are a hyperfusogenic line subcloned from XC cells.
- the 293/12 cell line is a 293 cell sub-line expressing eco- tropic receptor protein ATRC-1 (also known as MCAT1) (Ragheb et al., J. Virol. , Vol. 69, pgs. 7205-7215, (1995)).
- ATRC-1 also known as MCAT1
- Cells were maintained in D10: Dulbecco's modified essential medium, (Cell Culture Core Facility, USC) , 10% fetal calf serum (FCS) , 2 mM glu- tamine .
- Melittin was obtained from Sigma Chemical Co. (St. Louis, MO). Peptides were synthesized and HPLC-purified at the USC Norris Core Facility. Sequences were verified by mass spectroscopy . Peptide names indicate the first and the last residue number corresponding to the Moloney Murine Leukemia Virus (MoMuLV) env amino acid sequence. If the peptide has a residue different from the wild type MoMuLV env sequence, the wild type residue is listed followed by the number of the residue followed by the mutated amino acid (eg., 598-616 R609C) .
- MoMuLV Moloney Murine Leukemia Virus
- Liposomes were prepared from l-Palmitoyl-2-Oleoyl-sn-Glycero-3- (Phospho-rac-(l-glycerol) ) (POPG) and l-Palmitoyl-2-Oleoyl-sn- Glycero-3-Phosphocholine (POPC) (Avanti Polar Lipids Inc., Birming ⁇ ham, AL) .
- POPG l-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine
- POPC l-Palmitoyl-2-Oleoyl-sn- Glycero-3-Phosphocholine
- the lipid-chloroform solutions were mixed at a molar ra ⁇ tio of 1:3 (POPG:POPC), dried under N 2 and vacuum, sonicated, and taken through freeze-thaw cycles repeated a minimum of 5 times.
- the peptides for circular dichroism (CD) analysis were used at a final concentration of 50 UM.
- the stock solution of extruded liposomes at 50 mg/ l was used.
- the final peptide-to-lipid molar ratio was 1/100 in 20 mM Na 2 P0 4 buffer.
- CD spectra were obtained us ⁇ ing a Jasco J-600 CD spectrometer. Samples were scanned from 250 nm to 195 nm using cuvettes with a path length of 1 mm. Each result represents an average of 9 scans .
- Liposomes (see above) were made in 100 mM KCl, 10 mM Tris-HCl, pH 7.5. The external KCl was reduced to less than 0.1 mM by two passages through a PD10 col ⁇ umn (Pharmacia Biotech AB, Uppsala, Sweden) equilibrated in NaCl buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5). Potassium release from liposomes was detected by a K'-selective microelectrode (Micro- electrode Inc.) and recorded on VHS tape using a pulse-code modulator.
- a K'-selective microelectrode Micro- electrode Inc.
- Peptides were assayed at 10 ⁇ M and 30 UM.
- 500 ⁇ m diameter black membrane was formed from a 15 mg/ml solution of POPG /POPC (1/3) in n- heptane as described in Kagan et al . , Meth. Enzymol . , Vol. 235, pgs. 699-713 (1994).
- Membrane was kept at 50 mV in 10 mM KCl, 10 mM Tris-HCl, pH 7.5, and conductance was tested as a consequence of addition of 3 ⁇ M peptide.
- the envelope protein mutants were constructed in the ecotropic env expression vector pCEE * (MacKrell, et al . , J. Virol .. Vol. 70, pgs. 1768-1774 (1996)) using the oligonucleotide-directed in vitro mutagenesis system Quickchange (Stratagene, La Jolla, CA) .
- the name of the mutant reflects the amino acid position of the mutation with the wild type residue in one letter code on the left and the mutant residue on the right.
- An asterisk (*) represents a stop codon after the residue indicated.
- Chimeric env constructs containing either a melittin-like sequence, a hydrophilic sequence, or a random sequence were cloned into the CEE+ plasmid by overlap PCR oligonucleotide-directed in vi tro mutagenesis using the version 2.1 Amersham kit (Arlington Heights, IL) .
- the Notl /Nsil fragments from mutated plasmids were recloned into new CEE+ backbones and the cytoplasmic substitutions were verified by sequencing.
- the cytoplasmic env chimeras were introduced after 1598.
- the melittin-like sequence is: LKVLTTGLPAL ⁇ f- SWIstop (the italicized M resulted from a PCR error of lie to Met, because the sequence retained an amphiphilic character, it was used) .
- the hydrophilic sequence is: HNRKLQHNKDRRSstop (the native hydrophobic amino acids were substituted by hydrophilic residues) .
- the random sequence is: RFVNVNLRDYRFSDQSRLstop.
- NIH3T3 cells (lHlO 5 ) , 293T or 293/12 (lHlO 6 ) per 60 mm 2 plates were transfected according to the Ca(P0 4 ) 2 transfection protocol of 5'-3' Inc. (Boulder, CO) (day 1).
- 15 ⁇ g env expression plasmid was used.
- 10 ⁇ g of env MacKrell, et al . , (1996)
- 10 ⁇ g of pHIT 112 gag-pol
- 10 ⁇ g of pHIT 60 ( ⁇ -gal) (Soneoka, et al . , Nucl . Acids Res.
- FIG. 1 A A schematic representation of the MoMuLV env is shown in Fig. 1 A.
- a neural network program (Rost and Sander, Proteins , Vol. 19, pgs. 55- 72(1994)) predicts that in a hydrophobic environment the membrane proximal region 598-616 of the env cytoplasmic tail will fold into an ⁇ - helix.
- helical-wheel method for visualizing amphiphilic_ ⁇ -helixes suggests a helical nature for the mature cytoplasmic tail.
- the method predicts a distinct amphi- philicity for the membrane-proximal segment 598-616, with residues positioned on either a hydrophobic or on a hydrophilic side of the helix in agreement with their polar characteristics, with the notable exception of Arg 609 (Fig.l B, C) .
- the predicted amphiphilic ⁇ helix 598-616 extends three residues into the presumed viral membrane-spanning region and is preceded by a predicted turn sequence of Gly-Pro-Cys .
- the amphiphilicity ends at residue 616, which also corresponds to the cleavage site of the R peptide.
- the peptides 598-616 and 617-632 were evaluated by CD spectroscopy.
- the CD spectrum pro-ucked by the peptide 598-616 absorption of polarized light in an aqueous environment is characteristic of a random coil conformation
- peptide 598-616 becomes ⁇ -helical (Fig. 2 A, solid line) .
- the peptide 617-632 retained random coil conformation both in aqueous and in lipid environments
- 598-616 is nearly 60%. Similar ⁇ -helical properties have been observed for the lytic amphiphilic segments from HIV-1 env cytoplasmic tail and the active component of bee venom, melittin (Eisen- berg, et al . , Biopolymers, Vol. 29, pgs. 171-177 (1992)). Thus, the computer-predicted ⁇ -helical structure for the region 598-616 is confirmed for peptide 598-616 by CD analysis in the presence of membranes .
- topology and sequence-specific secondary structure of membrane-bound peptides and proteins can be determined from the acces- sibility of incorporated Rl side chains to collision with polar (NiEDDA) and non-polar (O 2 ) paramagnetic reagents in solution (Hub- bell and Altenbach, Curr. Opin. in Struct. Biol., Vol 4, pgs. 566- 573 (1994)).
- the accessibility is expressed by the quantity II, proportional to the collision frequency of the reagent with the nitroxide (Farahbakhsh, et al . , Photochem. & Photobiol .. Vol. 56, pgs. 1019-1033 (1992)).
- the topology parameter ⁇
- a Peptide name reflects first and last residue corresponding to the position in MoMuLV env. The position of a mutation is shown with the wild type residue followed by the site of the mutation and the identity of the mutant residue.
- peptide 598-616 with mutations at position 609 were tested in membrane destabilization assays.
- the Arg 609 Cys mutation in peptide 598-616 lowered the level of K * release by over 85% of the wild type peptide 598-616.
- peptide 598-616 Arg 609 Ala lost 75% of its activity.
- the peptide 598-616 with the double mutation Val 606 Arg / Arg 609 Val was made to reposition Arg by one helical turn, but to retain the positive charge on the hydrophobic side of the amphiphilic helix. This peptide is membrane-active, although at about 50% activity relative to the wild-type.
- planar lipid membrane data suggest a molecular mechanism for membrane destabilization. If the peptide 598-616 were to form pores, an equal stepwise increase in planar membrane conductance that does not result in membrane rupture would be expected. Such is seen in the incorporation of uniform ion channels of Borrelia Burgdorferi porin protein (Fig. 3, insert) (Lin, et al . , J. Biol. Chem.. Vol. 272, pgs. 44-47 (1997)). The peptide 598-616, however, causes a chaotic membrane disruption process that culminates in membrane rupture (Fig. 3). This result is more consistent with a series of monomeric peptides associating with the membrane rather than with multimeric channel formation.
- the end point fusion assay measures env-induced cell-to-cell fusion between env-transfected 293T cells and the ecotropic receptor expressing XC cells at 36 hrs post-transfection.
- the R-less (616*) envelope protein is the most fusogenic, over 2.5 times greater than the wild type envelope protein.
- the truncation at the presumed membrane spanning stop-transfer boundary at Arg 601 (601*) reduces envelope protein fusion activity to the level of the wild type envelope protein that retains the R peptide.
- the elimination of the remaining segment of the proposed ⁇ -helical structure in mutants 598*, 595*, and 595 Ser Arg* results in a dramatic decrease of fusion to near background levels.
- glycolipid-anchored envelope protein ectodomain (GLA ecto) and the tailless mutants except for the truncation retaining 8 residues of the membrane spanning region (578*) of the transmembrane subunit express on the cell surface (Appendix IB) .
- the glycolipid-anchored envelope protein ectodomain (GLA ecto) and the truncation retaining 8 residues of the membrane spanning region (578*) of the transmembrane subunit are fusion incompetent (Appendix IB, Figure 4A) .
- the other envelope protein with cytoplasmic truncations exhibit reduced fusion compared to the R-less env.
- the fusion kinetics of the 601* envelope protein are slightly faster than those of the wild type envelope protein, while rates of syncytia formation by the truncated envelope protein 595 Ser Arg* and the GLA ecto envelope protein are equal to the background.
- the syncytia formed by the 616* envelope protein at 24 to 30 hours post-transfection are 3 to 4 times more abundant than those formed by the wild type envelope protein.
- the rate of fusion indicates that removal of the region 598-616 affects envelope protein- mediated fusion adversely.
- cytoplasmic tail region of the envelope protein has been speculated to interact with the matrix (Freed, et al . , J. Virol. , Vol. 69, pgs. 1984-1989 (1996); Vzorov, et al., Virology, Vol. 221, pgs. 22-33 (1996)), the effect of cytoplasmic truncations on the efficiency of incorporation of the envelope protein into viral particles and on titers was assessed.
- Vi- rions were collected from the supernatant of 293T cells transfected with the env, ⁇ -gal , and the gag-pol expression plasmids, and analyzed for the level of envelope protein (SU gp 70 and TM pl5E) by Western Blot (Fig. 5 A, Appendix 1 B) .
- the incorporation of the R-less envelope protein (616*) is considerably less efficient than that of the wild-type envelope protein ( Figure 5A, Appendix IB) , and is reduced slightly more in the case of the 601* envelope protein.
- the 578* envelope protein is not detected in virions .
- Viral titer (Fig. 4A, white bars, Appendix IB) is reduced 10 times for the 616* envelope protein virions, and decreased 100 times for the 601*envelope protein.
- the titers for 598*, 595*, and 595 Ser Arg* envelope protein-containing particles are reduced by three to four orders of magnitude. No titer was detected for the particles with 578* or GLA ecto envelope protein.
- the progressive truncations of the envelope protein cytoplasmic tail correlate with the progressive decrease of envelope protein incorporation and a subsequent progressive reduction in titer.
- the Electrophysiological data indicate that the efficient membrane-destabilizing ability of peptide 598-616 depends on the presence of Arg 609 (Fig. 3), and the EPR data suggest that Arg 609 faces the membrane (Fig. 2 C) .
- Fig. 3 The Electrophysiological data indicate that the efficient membrane-destabilizing ability of peptide 598-616 depends on the presence of Arg 609 (Fig. 3), and the EPR data suggest that Arg 609 faces the membrane (Fig. 2 C) .
- Arg 609 mutant env proteins corresponding to the peptides assayed in vi tro, were made and assayed (Appendix IC) .
- the Arg 609 Cys env mutant was. «j ⁇ ot informative because it does not express efficiently.
- the efficiently expressed Arg 609 Ala env has one half the fusion activity, wild-type level of incorporation, and a normal titer.
- the MoMuLV envelope protein with a substitution of region 598-616 by a heterologous amphiphilic ⁇ -h ⁇ lix retains efficient fusogenicity
- the env expression plasmids were transfected transiently into NIH 3T3 cells because of the low fusogenicity of these cells compared with the 293T/XC co-culture system.
- the data are normalized to the fusogenicity of 616*, the wild type R-less envelope protein ( Figure 4C, Appendix IA) .
- the hydrophilic and the random-tail chimeras form syncytia inefficiently, 1% and 8% compared to the R-less wild type (616*) fusion activity.
- the fusogenicity of these chimeras in the 293T/XC co-culture assays also is reduced severely compared to both R-less and the wild type envelope protein (data not shown) .
- the Moloney/melittin chimeric envelope protein is at least as fusogenic as the 616* envelope protein.
- the data indicate that potent fusion activity of R-less Moloney envelope protein (616*) is reduced when the hypothesized amphiphilic membrane-proximal region is shortened (601*), but retained if replaced by a heterologous segment from an amphiphilic peptide.
- the efficiency of the envelope protein with cytoplasmic substitutions to mediate cell fusion was monitored by transient transfection of env constructs into NIH3T3 cells. Since in NIH3T3 cells there is no viral protease to cleave the R peptide, all of the chimeric env were engineered in the R-less form, to resemble the mature env cytoplasmic tail. All of the chimeric envelope protein constructs are expressed on the cell surface (Appendix 1 C) . Fusion of the R-less envelope protein was assayed by monitoring the formation of env-induced cell-to-cell fusion scored as syncytia in NIH3T3 due to the low fusogenicity of this cell line (Fig.
- the fusogenicity of envelope protein 601* is at least 4 fold lower than that of the envelope protein 616* or the Moloney/melittin chimeric envelope protein.
- the data indicate that potent fusion activity of R-less Moloney envelope protein (616*) is reduced when the hypothesized amphiphilic membrane-proximal region is shortened (601*), but retained if replaced by a heterologous segment from an amphiphilic peptide.
- the MoMuLV envelope protein with a substitution of region 598-616 with a heterologous amphiphilic ⁇ -helix efficiently incorporates envelope protein into virions and retains wild type transduction level
- Virions containing envelope protein constructs with cytoplasmic substitutions were produced in 293T cells as described and were tested for efficiency of incorporation into viral particles (Figure 5A, Appendix IA) .
- the Moloney/melittin envelope protein was incorporated efficiently into virions, while the incorporation of hydrophilic and random chimeric envelope protein was reduced. This result suggests that the secondary structure of the membrane-proximal region is important for incorporation.
- the envelope protein constructs with cytoplasmic substitutions next were tested for their ability to transduce NIH3T3 host cells.
- Virions with the Moloney/melittin tail had near wild type transduction levels (3X10 5 cfu/ml; Appendix 1 A).
- the presence of the hydrophilic or the random tail reduced titer by two orders of magnitude.
- successful replacement of the region 598-616 by a heterologous amphiphilic ⁇ -helix indicates that the functional role of the envelope protein membrane-proximal domain is influenced by its secondary structure rather than by a specific sequence.
- a conserved amphiphilic motif in envelope protein membrane-proximal regions identified by computational analysis The possibility that structural similarities exist among viral envelope protein membrane-proximal cytoplasmic tail regions was addressed computationally.
- the hallmark characteristic of an amphiphilic structure is its hydrophobic moment ( ⁇ ) . Domains with a high ⁇ value were calculated for a number of non-related viral envelope protein cytoplasmic sequences (Appendix 2, column C) . For most viruses analyzed (one exception shown is influenza HA) , the envelope protein cytoplasmic membrane-proximal region was calculated to have a high ⁇ .
- Appendix 2 includes the C-terminal HIV-1 cytoplasmic tail segment 1 ( ⁇ value of 2.21) previously calculated to have the second highest ⁇ value among all proteins in the data bank for 1989 (Eisenberg, et al . , 1990). Segment 1 of the HIV-1 tail, however, is membrane-distal and is not essential for virus entry; it is included here to serve as an amphiphilicity reference. As in other viruses analyzed, a high ⁇ also was identified for the HIV-1 envelope protein membrane-proximal cytoplasmic region. Because many lytic peptides are amphiphilic, it is relevant to note that the calculated envelope protein membrane-proximal ⁇ values often are higher than those in lytic peptides. Also shown for comparison in Appendix 2 is the ⁇ value of the lytic peptide melittin (1.23) .
- the melittin fragment used in the cytoplasmic substitution has a ⁇ value of 1.42.
- Proline and glycine may provide flexibility between the membrane-spanning and the membrane-proximal helices. Proline can contribute to the formation of the L-shaped structure between two helices (Efimov, 1992). Cysteine, if lipid modified (demonstrated for HIV-1, SIV, RSV, MPMV, MoMuLV, some HA isolates) , may serve as a protector against the disturbances at the tail reverberating into the ectodomain.
- SIV envelope protein was also suggested to be dependent on the envelope protein cytoplasmic domain (Vzorov, et al . , 1996). This conclusion is further supported by the SIV matrix structure (Rao, et al . , Nature, Vol. 378, pgs. 743-747 (1995)), the exposed side of which corresponds to the region affecting envelope protein incorporation. Current and previous (Januszeski et al . , 1997) results from progressive cytoplasmic truncations of the MoMuLV envelope protein cytoplasmic tail region also suggest that efficiency of envelope protein particle incorporation correlates with the integrity of the cytoplasmic tail region of the envelope protein.
- the membrane-proximal domain 598-616 is represented as connected flexibly to the membrane-spanning helix via Gly 595 and Pro 596.
- the CD data indicate that the peptide 598-616 is non- helical in the absence of a lipid-water interface, but currently no data is available on what the actual structure of the unprocessed cytoplasmic tail region may be.
- the domain 598-616 is represented as a helix prior to R peptide cleavage.
- the domain 598-616 is suggested to spiral up into the membrane, forming an amphiphilic ⁇ - helix parallel to the lipid bilayer.
- This burying of a helix is likely to create structural tension in between the two perpendicular helixes: the membrane-spanning (570-595) and the membrane- proximal (598-616) .
- Such tension may translate into a membrane disturbance at the base of the membrane-spanning domain, as well as along the length of the now membrane-embedded helix 598-616.
- This suggested burying of the amphiphilic helix into the membrane with Arg 609 oriented towards the membrane is proposed to cause fusion- potentiating destabilization of the viral membrane inner leaflet.
- Arg 609 is required for the activity of the isolated peptide, as measured by the electrophysiologic assays, the presence of Arg 609 is not necessary, although it potentiates fusion in the context of the whole envelope protein.
- the data indicate that the truncations that eliminate Arg 609 or mutate it do not eliminate fusion, although they do reduce it from the maximum.
- a caveat to interpretation of data based on any cell-to-cell fusion assays is that the mechanism of syncytia formation may not be identical to virus-to-cell fusion. This caution also pertains to the correlation of the fusion and the transduction data obtained in different cell assays.
- Arg 609 mutants and the other cytoplasmic tail region mutants are being analyzed further with the attention to the hypothesis of Martin et al . , J. Virol., Vol. 70, pgs. 298-304 (1996) that a fusion peptide is active when it enters the membrane at an oblique angle.
- the successful functional substitution with the melittin segment indicates the importance of the secondary structure of the domain 598-616 for its function.
- the melittin-like cytoplasmic domain may be argued to function not by substituting an analogous function, but merely by stabilizing the ectodomain; however, envelope protein chimeras with random and hydrophilic tails are not fusogenic.
- previous saturation mutagenesis data of the membrane-proximal region (Januszeski et al . , 1997) indicate that mutations that disrupt amphiphilicity and reduce hydrophobicity of the membrane-proximal region have a negative effect on fusion.
- current data suggest that the structure of the membrane- proximal domain determines its function.
- the destabilization of the viral membrane above the R-less tail may be sufficient to bring fusion of host and viral membranes to completion.
- the hypothetical structure is modeled as a trimer (Fig. 6B) based on the crystallography of the MoMuLV ectodomain TM segment
- the current literature point to the possible interaction of the envelope protein cytoplasmic tail region with the viral core.
- the data on the cytoplasmic tail region truncations further indicate that the presence of the R peptide has a positive effect on incorporation, since its removal decreases envelope protein incorporation.
- removing of the membrane-proximal region results in further significant decrease of envelope protein incorporation.
- structurally solved retroviral ectodomain segments and matrices are both trimers, and the overall architecture of the three resolved viral matrices of HIV-1, SIV, and BLV (Hill, et al., Proc. Nat. Acad. Sci. , Vol. 93, pgs. 3099-3104 (1996); Rao, et al .
- the surfaces of both trimers are outlined by three ⁇ -helixes forming an equilateral triangle with similar architecture and dimensions.
- the MoMuLV envelope protein sub-cytoplasmic trimer has a side of 67 A and the SIV matrix measures at 68 ⁇ 8 A (Rao, et al . , 1995) .
- At the corner of each membrane-parallel helix in the HIV-1 matrix crystal there is a long protruding helix which may serve as a support for the membrane-distal amphiphilic lentiviral tail.
- the proposed trimeric unit (Fig.
- Peptides corresponding to the cytoplasmic tail region of the Moloney Murine Leukemia Virus envelope protein were synthesized according to the procedure described in Example 1.
- Unilamellar liposomes formed from (i) POPC; (ii) POPC and POPG at a molar ratio of POPC: POPG of 3:1, or (iii) POPC and POPG at a molar ratio of POPC: POPG of 1 : 1 were prepared in lOOmM KCl according to the procedure described in Example 1.
- Peptide-induced K+ release from the liposomes also was detected according to the procedure described in Example 1. All measurements were carried out at 22°C. The results are given in Table III below.
- Triton X - ⁇ oo 100 100 100
- the surface envelope protein expression measured by indirect im- munofluorescense using env-specific antibody on env-transfected 293T cells.
- the relative fusion efficiency was determined as syncytia formation in env-transfected NIH3T3 cells (Table A column only) or as the ability to induce syncytia formation in XC6 cell co-cultured with env-transfected 293T cells.
- d' A mutant ability to transduce host NIH3T3 cells is shown as relative ⁇ -gal titer. Same supernatant used for titers were used for Western Blots. The average wild type envelope protein titer was 4X10 5 cfu/ml for (A) and 1X10 6 cfu/ml for (B) and (C) .
- HIV1 2 .21 P17, R21, 151 NA segl '" 128-151 G23
- viruses and proteins Abbreviations of viruses and proteins: avian leukosis - ALV, bovine leukemia - BLV, equine infectious anemia - EIA, feline immu- nideficiency - FIV, hepatitis C-HEPC, human immunodeficiency - HIV, human T cell leukemia - HTLV, human respiratory syncytial - hRSV, influenza - INF, murine mammary tumor - MMTV, Mason Pfizer monkey - MPMV, Rous Sarcoma - RSV, parainfluenza - PINF, spleen necrosis - SNV, vesicular stomatitis - VSV, simian sarcoma - Sim Src V-HLB, Herpes Simplex virus glycoprotein H - HSV gH, Simian virus - SV5.
Abstract
Description
Claims
Priority Applications (8)
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JP2000559138A JP2003533165A (en) | 1998-07-09 | 1999-07-08 | An isolated amphipathic peptide from the cytoplasmic end of the viral envelope protein |
CA002333855A CA2333855A1 (en) | 1998-07-09 | 1999-07-08 | Isolated amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins |
EP99926703A EP1093519A2 (en) | 1998-07-09 | 1999-07-08 | Amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins |
AU43869/99A AU4386999A (en) | 1998-07-09 | 1999-07-08 | Isolated amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins |
IL14063099A IL140630A0 (en) | 1998-07-09 | 1999-07-08 | Isolated amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins |
US09/756,250 US20020058020A1 (en) | 1999-07-08 | 2001-01-08 | Isolated amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins |
US10/304,494 US20040022799A1 (en) | 1998-07-09 | 2002-11-25 | Isolated amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins |
AU2003235023A AU2003235023A1 (en) | 1998-07-09 | 2003-08-13 | Isolated amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins |
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US11254498A | 1998-07-09 | 1998-07-09 | |
US09/112,544 | 1998-07-09 |
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US09/756,250 Continuation US20020058020A1 (en) | 1998-07-09 | 2001-01-08 | Isolated amphiphilic peptides derived from the cytoplasmic tail of viral envelope proteins |
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EP (1) | EP1093519A2 (en) |
JP (1) | JP2003533165A (en) |
AU (2) | AU4386999A (en) |
CA (1) | CA2333855A1 (en) |
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Cited By (2)
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WO2001012235A2 (en) * | 1999-08-19 | 2001-02-22 | University Of Southern California | Targeted artificial gene delivery |
US7785903B2 (en) | 2004-04-09 | 2010-08-31 | Genentech, Inc. | Variable domain library and uses |
Citations (4)
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WO1993005147A1 (en) * | 1991-08-30 | 1993-03-18 | The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Defective interfering hiv particles with chimeric cd4-env |
WO1994011524A1 (en) * | 1992-11-09 | 1994-05-26 | The United States Government As Represented By The Secretary Of The Department Of Health And Human Services | Targetable vector particles |
WO1996018957A1 (en) * | 1994-12-14 | 1996-06-20 | The Trustees Of Columbia University In The City Of New York | A method for predicting protein structure |
WO1997033908A1 (en) * | 1996-03-13 | 1997-09-18 | Commonwealth Scientific And Industrial Research Organisation | Lytic peptides |
-
1999
- 1999-07-08 EP EP99926703A patent/EP1093519A2/en not_active Withdrawn
- 1999-07-08 IL IL14063099A patent/IL140630A0/en unknown
- 1999-07-08 AU AU43869/99A patent/AU4386999A/en not_active Abandoned
- 1999-07-08 CA CA002333855A patent/CA2333855A1/en not_active Abandoned
- 1999-07-08 JP JP2000559138A patent/JP2003533165A/en active Pending
- 1999-07-08 WO PCT/IB1999/001261 patent/WO2000002909A2/en not_active Application Discontinuation
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2003
- 2003-08-13 AU AU2003235023A patent/AU2003235023A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993005147A1 (en) * | 1991-08-30 | 1993-03-18 | The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Defective interfering hiv particles with chimeric cd4-env |
WO1994011524A1 (en) * | 1992-11-09 | 1994-05-26 | The United States Government As Represented By The Secretary Of The Department Of Health And Human Services | Targetable vector particles |
WO1996018957A1 (en) * | 1994-12-14 | 1996-06-20 | The Trustees Of Columbia University In The City Of New York | A method for predicting protein structure |
WO1997033908A1 (en) * | 1996-03-13 | 1997-09-18 | Commonwealth Scientific And Industrial Research Organisation | Lytic peptides |
Non-Patent Citations (6)
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EISENBERG D AND WESSON M: "The most highly amphiphilic alpha-helices include two amino acid segments in human immunodeficiency virus glycoprotein 41" BIOPOLYMERS, vol. 29, no. 1, 1992, pages 171-177, XP000874043 cited in the application * |
JANUSZESKI M.M ET AL., : "Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein" JOURNAL OF VIROLOGY, vol. 71, no. 5, May 1997 (1997-05), pages 3613-3619, XP002130560 cited in the application * |
MARTIN I ET AL., : "Lipid membrane fusion induced by the human immunodeficiency virus type 1 gp41 N-terminal extremity is determind by its orientation in the lipid bilayer" JOURNAL OF VIROLOGY, vol. 70, no. 1, January 1996 (1996-01), pages 298-304, XP002130559 cited in the application * |
RAGHEB JA AND ANDERSON WF: "pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry" JOURNAL OF VIROLOGY, vol. 68, no. 5, May 1994 (1994-05), pages 3220-3231, XP000874027 cited in the application * |
SABERWAL G AND NAGARAJ R: "Cell-lytic and antibacterial peptides that act by perturbing the barrier function of membranes: facets of their conformational features, structure-function correlations and membrane-perturbing abilities" BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1197, no. 2, 1994, pages 109-131, XP000878719 cited in the application * |
VZOROV A.N. ET AL., : "Assembly and release o SIV Env proteins with full-length or truncated cytoplasmic domains" VIROLOGY, vol. 221, 1996, pages 22-33, XP002921436 cited in the application * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001012235A2 (en) * | 1999-08-19 | 2001-02-22 | University Of Southern California | Targeted artificial gene delivery |
WO2001012235A3 (en) * | 1999-08-19 | 2001-05-25 | Univ Southern California | Targeted artificial gene delivery |
JP2003507348A (en) * | 1999-08-19 | 2003-02-25 | ユニバーシティ オブ サザン カリフォルニア | Targeted artificial gene delivery |
US7785903B2 (en) | 2004-04-09 | 2010-08-31 | Genentech, Inc. | Variable domain library and uses |
Also Published As
Publication number | Publication date |
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CA2333855A1 (en) | 2000-01-20 |
AU2003235023A1 (en) | 2003-09-11 |
WO2000002909A3 (en) | 2000-05-04 |
EP1093519A2 (en) | 2001-04-25 |
JP2003533165A (en) | 2003-11-11 |
AU4386999A (en) | 2000-02-01 |
IL140630A0 (en) | 2002-02-10 |
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