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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/542—Mucosal route oral/gastrointestinal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
• • 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
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—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
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• Dispersed aggregates of non-encapsulated lymphoid tissue are often localized to the submucosal areas of the gastrointestinal, respiratory and urogenital tracts. These tracts are a main means of entry into the body by foreign microorganisms.
• Secretory immunoglobulin A (IgA) is an antibody capable of crossing mucosal membranes and protecting them against invasion by pathogens. Mucosal lymphoid tissue thus plays an important role in the local immune response which occurs at mucosal surfaces.
• mucosal epithelial cells regardless of whether they express the cell surface CD4 receptor used by the HIV to enter T-cells, macrophages and Langerhans cells, can be latently infected by HIV (Fantini et al., (1992) J. Virol . , 66: 5805; Fantini et al . , (1991) Virology, 185: 904) .
• the receptor(s) for HIV entry into mucosal intestinal epithelial cells appear to be glycolipids (Yahi et al. , (1992) J. Virol . , 66: 4848) , there is no information regarding the HIV epitope(s) which mediates attachment to these cells. Such knowledge would be of paramount importance since this epitope(s) would be an ideal target against which the local mucosal immune system could act to prevent the mucosal entry of HIV.
• CT cholera toxin
• Vibrio chol era is among the strongest mucosal immunogens known.
• CT binds strongly to a glycosphingolipid called ganglioside GM1 on mucosal cell surfaces using its B subunit.
• Mucosal administration of minute amounts of antigens covalently linked to the B subunit (CTB) has been shown to elicit vigorous mucosal as well as extramucosal immune responses in experimental animals including nonhuman primates (Czerkinsky et al., (1989) Infect . Immun .
• One embodiment of the present invention is a method for inhibiting the infection of mucosal cells by HIV-1 by administering a vaccine to the mucosa, thereby delivering to the mucosa a peptide of HIV-1 gpl20 having from about 10 to about 50 amino acids, whereby antibodies against the peptide are generated in the mucosa, the peptide being selected such that the antibodies inhibit infection of HIV-1 in mucosal epithelial cells.
• the peptide includes an epitope effective to generate mucosal production of antibodies that inhibit infection of mucosal cells by HIV-1, the peptide consisting essentially of SEQ ID NOS: 9, 10, 11, 12, or 13.
• the vaccine further includes an agent for enhancing delivery of the peptide to the mucosa.
• the agent is a mucosal binding protein; most preferably, it is either the binding subunit of cholera toxin or that of E. coli heat labile enterotoxin.
• the invention also provides that the peptide and the mucosal binding protein are bound together to form a chimeric protein which may advantageously be the expression product of recombinant DNA.
• the agent is a lipid.
• the lipid is in the form of a lipid vesicle.
• the administering step comprises administering to the mucosa a polynucleotide operably encoding the peptide, whereby the peptide is produced by cells of the mucosa.
• a further embodiment of the invention provides a vaccine for inhibiting the infection of mucosal cells by HIV-1, comprising a 10 to 50 amino acid peptide of HIV-1 gpl20 having an epitope selected such that antibodies against this epitope inhibit the infection of mucosal epithelial cells by HIV-1, and a compound or structure associated with the peptide for facilitating delivery of the peptide to the mucosa.
• this peptide consists essentially of SEQ ID NO 9, 10, 11, 12 or 13 and the compound or structure is a lipid vesicle.
• the compound or structure is a mucosal binding protein.
• the binding protein is a cholera toxin protein which may advantageously be the binding subunit.
• the binding protein is the binding subunit of E. coli heat labile enterotoxin.
• the present invention discloses the identification of synthetic peptides derived from the sequence of the envelope glycoprotein gpl20 of HIV-1. These peptides were used to generate neutralizing antibodies which inhibited infection of transformed human vaginal and colorectal cell lines in vi tro . These peptides will induce the production of a localized mucosal immune response, generating antibodies able to neutralize infection of human colorectal and vaginal epithelial cells by HIV-1.
• the peptides are set forth herein as SEQ ID NOS: 9-13. In one aspect of the invention, one or more of the peptides of SEQ ID NO: 9, 10, 11, 12, and 13 is used to generate antibodies.
• These antibodies can be generated in any conventional manner, including by intramuscular, intraperitoneal, subcutaneous, or mucosal administration to an animal. Generation of both monoclonal and polyclonal antibodies are contemplated. These antibodies are then used to prevent infection of cells of the mucosal epithelium by providing the antibodies in association with the mucosal cells and then challenging the cells with HIV-1. The antibodies inhibit or prevent binding of the virus to the cells, and thereby inhibit or prevent infection of the cells by the virus.
• the antibodies can be exogenous or endogenous antibodies, and the cells can be in vi tro or in vivo .
• the antibodies are typically generated in laboratory or domestic animals or are monoclonal antibodies. More importantly, it provides a valuable tool for analyzing the mechanism and structure involved in that binding.
• the antibodies are preferably endogenous mucosal antibodies that have been generated by administering one or more of the peptides of SEQ ID NOs 9-13 to the animal in which the cells are located. Mucosal vaccination, as described below, is particularly preferred. However, exogenous antibodies may also be administered to the animal to inhibit HIV-1 infection of mucosal cells. In all of the treatments described herein, the mucosal cells are preferably of human origin.
• the peptides of the present invention can be utilized alone or in combination and can also be uncoupled or coupled to other epithelial cell binding proteins including CT, CTB and the binding subunit of E. coli heat labile enterotoxin.
• the peptides may be coupled by either chemical or recombinant means.
• DNA encoding the peptides can be joined to DNA encoding cholera toxin, or its B subunit, by well known methods, inserted into a eukaryotic expression vector and delivered to epithelial cells using lipid vesicles or lamellar structures.
• peptide-CT, CTB or enterotoxin conjugates in vivo will then elicit a localized mucosal immune response and will protect against subsequent infection by HIV.
• mucosal epithelial cell binding proteins such as cholera toxin
• mucosal epithelial cell binding proteins will advantageously increase the efficiency of entry of peptides into these cells. Since the B subunit of the cholera toxin A-B dimer is responsible for binding to cell surface receptors, a peptide- CTB conjugate will also bind efficiently to epithelial cells.
• the literature also reports methods for forming compositions of immunogenic peptides and other gut binding proteins (Wenneras et al. , (1990) FEMS Microbiol . Lett .
• peptides are also useful in the production of monoclonal and polyclonal antibodies. These antibodies have a distinct neutralizing effect on HIV-1. These peptides, either alone or after coupling to CT or other molecules, maybe administered orally, rectally, vaginally, or in a combination of these routes in an amount sufficient to generate a mucosal antibody response sufficient to inhibit HIV-1 entry into the mucosal epithelial cells.
• the amounts of peptides used will depend on their pharmaceutical formulation and the site and route of delivery; however, for an adult human, a suitable immunogenic amount of peptide is generally between about 50 ⁇ g and about 1 mg, administered one to four times over a period of two weeks to one year or longer.
• the peptides, peptide-binding protein conjugates, and other compositions of the present invention can be administered orally to generate a localized gastrointestinal mucosal immune response or intravaginally or intrarectally to produce a localized mucosal immune response in these areas prone to viral entry by sexual contact.
• These peptides can be administered in unit dosage in an amount necessary to produce localized mucosal immunity against HIV-1 infection.
• compositions envisioned for oral administration include tablets, capsules, liquids, and the like and those contemplated for intravaginal or intrarectal administration include injectable carriers, suppositories, ointments, gels, creams, foams, sprays, dispersions, suspensions, pastes and the like in an amount from about 10 ⁇ g to about 10 mg or more.
• injectable carriers suppositories, ointments, gels, creams, foams, sprays, dispersions, suspensions, pastes and the like in an amount from about 10 ⁇ g to about 10 mg or more.
• These preparations can be in any suitable form, and generally comprise the active ingredient in combination with any of the well known pharmaceutically acceptable carriers.
• the preparations may further advantageously include preservatives, antibacterials, antifungals, antioxidants, osmotic agents, and similar materials in composition and quantity as is conventional.
• Remington's Pharmaceutical Sciences 15th Ed., Mack Publishing Co., Easton, PA (1975) .
• peptides or conjugates are able to be directly taken up by the epithelial cells lining these areas. These peptides and conjugates may advantageously be enclosed in liposomes to facilitate delivery of these agents to cells.
• Direct injection of the peptides or peptide-binding protein conjugates, either alone or in combination with lipid vesicles or other lamellar structures, into the mucosal endothelium in a similar dose range is also envisioned as a method of eliciting an anti-HIV response in these tissues.
• Example 1 Susceptibility of colorectal and vaginal epithelial cells to infection by HIV-1
• HIV-1 infectious virus stocks of HTLV-IIIB-infected H9 T cell lymphoma cells (ATCC HTB-176) (Popovic et al . , (1984) Science, 224: 497-500) were used in the following experiments.
• the cells were maintained in RPMI-1640 medium containing 20% fetal calf serum (FCS) , 100 units/ml penicillin and 100 ⁇ g/ml streptomycin.
• FCS fetal calf serum
• Virus stocks were prepared using well known procedures and frozen at -90°C.
• Endpoint titration of the HTLV-IIIB isolate of HIV-1 in two clones of transformed vaginal epithelial cells (Hs 760 T and Hs 769.Vg cells; ATCC CRL-7491 and 7499, respectively) and 12 subclones of colon adenocarcinoma HT-29 cells (ATCC HTB-38) were performed by inoculation of respective cell lines with serial 10-fold dilutions of virus with 100 ⁇ l/well (ranging from 1 TCID 50 /cell to 0.00001 TCID S0 /cell) in 24-well plates
• DMEM Modified Eagle's Medium
• FCS fetal calf serum
• HTLV-IIIB-infected H9 cells (10 6 cells) in H9 maintenance medium were added to each well and cocultured with epithelial cells for 24 hours.
• the H9 cultures were microscopically followed for 7 days for the presence of HIV-induced syncytium formation and p24 antigen production using an ELISA able to detect as little as 100 pg p24/ml) .
• the vaginal cell lines Hs 760.T and Hs 769.Vg were permissive.
• Viral infection of Hs 760.T was detected by coculture at a high multiplicity (1 TCID 50 /cell) and 6 of the HT-29 colon cell clones were permissive at multiplicities ranging from 0.1-0.01 TCID 50 /cell. Of these clones, cloOne L20 was chosen for further study.
• HTLV-IIIB Susceptibility of colorectal and vaginal epithelial cells to infection by HIV-1
• Epithelial cells were harvested seven days post-infection and DNA was extracted (Sambrook et al . , (1989) Mol ecular Cloning, second edition, Cold Spring Harbor Laboratory Press, 2: 9.16-9.19) .
• Primers specific for the HIV-1 env gene (5'- GTAACGCACAGTTTTAATTGTGGAGGGGAA-3' ; SEQ ID NO: 1) and (5'- CCTCATATTTCCTCCTCCAGGTCT-3' ; SEQ ID NO: 2) were used for detection of proviral DNA.
• DNA 200 ng
• the reaction mixture consisted of 10 ⁇ l of lOx PCR buffer (Promega, Madison, WI) , 1.5 mM MgCl 2 , 20 pmol primers, 0.125 mM dNTPs, 5 ⁇ Ci ⁇ .- 32 P-dCTP and 0.5 units Taq DNA polymerase (Promega) .
• the amplification was for 35 cycles and included denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min.
• One-tenth of the final reaction mixture was analyzed by electrophoresis on 5% polyacrylamide gels. The gels were dried and exposed to X-ray film (X-OMAT; Eastman Kodak, Rochester, NY) for 13-16 hours using an intensifying screen.
• HIV copy number the number of HIV genomes
• two-fold serial dilutions of DNA isolated from ACH-2 cells which contain one proviral copy per cell (Clouse et al . , (1989) J. Immunol . , 142: 431-438; Seshamma et al . , (1992) J. Virol . Methods , 40: 331-346; Graziosi et al . , (1993) Proc . Na tl . Acad . Sci . U. S. A . , 90: 6405-6409) .
• the total amount of DNA in each dilution was normalized to 200 ng using DNA extracted from H9 cells and PCR was performed as above.
• HIV copy number was estimated by comparison of the intensities of the amplified bands. PCR analysis using a pair of human ⁇ - actin primers was performed in parallel as 'an internal standard.
• Example 3 Detection of HIV RNA expression by RT-PCR Epithelial cells were harvested 7 days post-infection and total RNA was extracted by the RNAzol method (Biotex Laboratories, Houston, TX) . For each sample, 500 ng of total RNA was incubated with 10 units RNase-free DNase I (Boehringer Mannheim, Mannheim, Germany) at 37°C for 1 hour. Samples were then heated to 80°C for 10 min to degrade the DNase.
• cDNA was synthesized in a reverse transcriptase (RT) reaction with 10 pmol downstream PCR primer (described below) , 0.625 mM dNTPs, 5 x reaction buffer (Promega) and 200 units Moloney murine leukemia virus RT (Promega) to a final volume of 20 ⁇ l .
• the mixture was incubated at 37°C for 1 hour.
• the cDNA was amplified for 30 cycles by PCR as described in Example 3.
• the primers used to detect HIV-1 regulatory RNA were as follows: 5' -GAAGAAGCGGAGACAGCGACG-3' (SEQ ID NO: 3) 5' -GGCCTGTCGGGTCCCCTCG-3' (SEQ ID NO: 4)
• RNA without reverse transcriptase was also amplified by PCR to demonstrate that the amplified fragments were from HIV cDNA, not from contamination of HIV DNA.
• the primers for the first PCR were as follows: 5' -GAAGAAGAGATAGTAATTAGATCT-3' (SEQ ID NO: 7)
• the primers used for the second (nested) PCR were SEQ ID NO: 7 and SEQ ID NO: 2 .
• One-tenth of the first PCR product was added to the second PCR reaction.
• the PCR conditions were as described in Example 3, except that 40 cycles of amplification were performed.
• One-tenth of the final reaction mixture was analyzed by electrophoresis on 2% agarose gels and stained with ethidium bromide.
• RNA samples without RT were also amplified by the nested primers as a test for DNA contamination.
• DNA content and RNA expression in HIV-1 infected epithelial cells is shown in Table 2. Approximately 1% of HT29 L20 cells are infected with HIV-
• HIV-infected HT29 L20 cells if HIV-infected cells contain 1 copy of proviral DNA per cell.
• expression of regulatory RNA in HIV-1 infected HT29 L20 cells is lower than that in HIV-1 infected H9 cells and ACH-2 cells. Expression of structural RNA is barely detectable.
• HIV-1 HTLV-IIIB
• HTLV-IIIB HIV-1 (HTLV-IIIB) RNA expression in colorectal and vaginal epithelial cells
• Hyperimmune sera was isolated from monkeys immunized with the five peptides derived from the gpl20 sequence listed below (Table 3) .
• Solid phase peptide synthesis was performed using an Applied Biosystems (Foster City, CA) 430A peptide synthesizer. An amino-terminal cysteine residue was added to each peptide to facilitate coupling to a carrier protein. Peptides were covalently coupled to ovalbumin, grade V (Sigma, St. Louis, MO) at an approximate 10:1 (peptide:ovalbumin) molar ratio using N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP; Pharmacia, Uppsala, Sweden) .
• SPDP N-succinimidyl 3- (2-pyridyldithio) propionate
• Stock virus was diluted to 10 4 TCID 50 for neutralization in colon cells and used undiluted (10 6 TCID 50 ) for neutralization in vaginal cells and mixed with serial four fold dilutions of heat-inactivated monkey sera starting at 1:5. The monkey sera were used at a final dilution of 1:10 or 1:20. The guinea pig hyperimmune serum served as a positive control. After incubation for 2 hours at 37°C, the serum virus mixture was incubated with the epithelial cells for 2 hours at 37°C. The cells were washed twice with medium and supplemented with 1.5 ml of respective maintenance medium/well. Seven days after infection the cells were washed five times and treated with 0.1% trypsin at 37°C for 5 minutes.
• H9 cells (10 6 ) were added to each well and cocultures were monitored for 7 days for syncytia formation and presence of p24 antigen.
• Results for HS 760.T cells, Hs769 cells and HT-29 L20 cells are indicated in Tables 4/5, 6, and 7, respectively, and are expressed as mean neutralization titers, defined as the reciprocal of the serum dilution that reduced the p24 antigen by at least 90%.
• the HIV-1 copy number is also shown for HIV-1 infected HS 760.T cells (Tables 4 and 5) .
• HIV-1 HTLV-IIIB
• Hs 760.T cells Neutralization of HIV-1 (HTLV-IIIB) infectivity in Hs 760.T cells by monkey hyperimmune sera against gpl20 peptides.
• HIV-1 HTLV-IIIB
• Hs 760.T cells Neutralization of HIV-1 (HTLV-IIIB) infectivity in Hs 760.T cells by guinea pig ant ⁇ -gpl20 serum and monkey hyperimmune sera against gpl20 peptides.
• HIV-1 DNA neutralization assayed (copy/SxlCcells) by cocultivation
• HIV-1 HTLV-IIIB
• HS 769.Vg cells Neutralization of HIV-1 (HTLV-IIIB) infectivity in HS 769.Vg cells by guinea pig and monkey hyperimmune sera against gpl20.
• Guinea pig anti crpl20 dilution HIV-1 copy number (copy/10 4 cells) pre-immune post-immune x40 500 ⁇ 12.5 xl60 250 100 x640 250 100
• HIV-1 HIV-1
• HTLV-IIIB Neutralization of HIV-1 (HTLV-IIIB) infectivity in HT-29 L20 cells by guinea pig anti-gp 120 serum and monkey hyperimmune sera against gpl20 peptides.
• Serum Pre-immune Post-immune Post-immune Guinea pig anti-gpl20 + gpl20-l to gpl20-ll ND (aa 1-164) gpl20-12 (aa 152-176) gpl20-13 to gpl20-14 ND (aa 165-205) gpl20-15 (aa 193-218) gpl20-16 (aa 206-230) gpl20-17 to gpl20-18 ND (aa 219-257) gpl20-19 ND ND ND (aa 248-269) gpl20-20 to gpl20-23 ND (aa 258-320) gpl20-24 (aa 307-330) gpl20-25 to gpl20-40 ND (aa 321-511) mixture of gpl20- ND (12+15+16+19+24)
• DNA corresponding to peptides having the sequence of SEQ ID NO: 9-13 is linked to DNA encoding the B subunit of cholera toxin by standard methods of molecular biology.
• the resulting chimeric construct is placed in a commercially available eukaryotic expression vector such as pGEX (Pharmacia, Piscataway, NJ) containing the appropriate translation initiation and termination signals.
• pGEX Pulposia, Piscataway, NJ
• This construct is then incorporated into a lipid vesicle by methods well known in the art.
• the lipid vesicle is then formulated into a foam or suppository composition by well known pharmacolological preparation methods and administered vaginally and/or rectally to humans at high risk for HIV infection.
• the dose range administered is in the range of from about 10 ⁇ g to 10 mg.
• the administration is repeated at two week intervals for a total of three administrations.
• the presence of anti-HIV antibodies in the vaginal and rectal mucosa is assayed by isolating protein from vaginal secretions and feces (which contains cells shed from the vaginal and rectal epithelium, respectively) and performing a p24 ELISA to determine whether any antibodies are present.
• These antibodies can then be used in HIV-1 virus neutralization assays (Vahlne et al. , (1991) Proc . Na tl . Acad . Sci . U. S. A . , 88: 10744-10748) .
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• SEQUENCE DESCRIPTION SEQ ID NO : 1 : GTAACGCACA GTTTTAATTG TGGAGGGGAA 30
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO

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