WO2017004130A1 - Dégradation ou neutralisation d'apobec3b par le virus d'immunodéficience simienne vif - Google Patents

Dégradation ou neutralisation d'apobec3b par le virus d'immunodéficience simienne vif Download PDF

Info

Publication number
WO2017004130A1
WO2017004130A1 PCT/US2016/039973 US2016039973W WO2017004130A1 WO 2017004130 A1 WO2017004130 A1 WO 2017004130A1 US 2016039973 W US2016039973 W US 2016039973W WO 2017004130 A1 WO2017004130 A1 WO 2017004130A1
Authority
WO
WIPO (PCT)
Prior art keywords
vif
cell
polynucleotide
siv
sivmac239
Prior art date
Application number
PCT/US2016/039973
Other languages
English (en)
Inventor
Reuben S. Harris
Allison LAND
Original Assignee
Regents Of The University Of Minnesota
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regents Of The University Of Minnesota filed Critical Regents Of The University Of Minnesota
Publication of WO2017004130A1 publication Critical patent/WO2017004130A1/fr

Links

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
    • C07K14/08—RNA viruses
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
    • 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
    • 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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • 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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • 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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • APOBEC3B is a member of a family of APOBEC3 (A3) proteins that deaminate DNA cytosine bases to produce the pro-mutagenic lesion, uracil.
  • APOBEC3B is a source of mutations in breast, lung, head/neck, bladder, cervical, and several other types of cancer.
  • this disclosure describes a polynucleotide that includes SEQ ID NO:2.
  • this disclosure describes a method that includes providing tumor cells, providing a polynucleotide encoding at least a portion of a simian immunodeficiency virus (SIV) Vif gene, and introducing the polynucleotide into the tumor cells.
  • SIV simian immunodeficiency virus
  • the polynucleotide encodes a protein having the amino acid sequence of SEQ ID NO:3. In some of these embodiments, the polynucleotide includes SEQ ID NO:2. In some embodiments, the polynucleotide encodes a sequence that includes a conserved SLQ tri-residue peptide motif.
  • the polynucleotide encodes a sequence that includes a conserved TLQ tri-residue peptide motif.
  • the polynucleotide encodes at least a portion of a simian
  • the SIV Vif protein can include SIVmac239 Vif, SIVsmCFU212 Vif, SIVsmPG Vif, SIVsmPBj Vif, SIVsmE041 Vif, SIVstm Vif, SIVmacl42 Vif, SIVmfal86 Vif, SIVmne07 Vif, or SIVsmE543 Vif.
  • the polynucleotide is codon-optimized for expression in a human cell.
  • this disclosure describes a method that includes introducing into a cell a polynucleotide encoding a simian immunodeficiency virus (SIV) Vif, wherein expression of the SIV Vif in the cell results in modulation of the functional activity of APOBEC3B in the cell.
  • SIV simian immunodeficiency virus
  • the cell is a cancer cell. In some of these embodiments, the cell is a breast cancer cell.
  • the cell is a precancerous cell.
  • the cell is a human papilloma virus (HPV)-infected cervical cell.
  • HPV human papilloma virus
  • the cell is a human cell.
  • the polynucleotide encodes a protein comprising SEQ ID NO:3. In some of these embodiments, the polynucleotide includes SEQ ID NO:2.
  • the polynucleotide encodes a conserved SLQ tri-residue peptide motif or a conserved TLQ tri-residue peptide motif.
  • the polynucleotide encodes at least a portion of a simian
  • the SIV Vif protein can include SIVmac239 Vif, SIVsmCFU212 Vif, SIVsmPG Vif, SIVsmPBj Vif, SIVsmE041 Vif, SIVstm Vif, SIVmacl42 Vif, SIVmfal86 Vif, SIVmne07 Vif, or SIVsmE543 Vif.
  • the polynucleotide is codon-optimized for expression in a human cell.
  • the method further includes introducing into the cell a vector that includes the polynucleotide.
  • the vector is a viral vector.
  • the polynucleotide is RNA. In some of these embodiments, the RNA is delivered in a small particle or a viral particle.
  • the term "gene” refers to a region of a deoxyribonucleic acid that encodes a protein.
  • a gene can include certain non-coding sequences (e.g., one or more introns) or may be intronless, whether natively intronless or derived from cDNA.
  • the transfer of a gene from one organism to another can include the transfer of expression control sequences native to the gene being transferred. Alternatively, the gene may be placed under the control of heterologous expression control sequences.
  • operably linked refers to a functional linkage between a first nucleic acid sequence and second nucleic acid sequence in such a manner as to allow general functions.
  • a nucleic acid sequence coding for a target protein or RNA may be operably linked to a nucleic acid expression control sequence, in such a manner that the expression control sequence affects expression of the coding nucleic acid sequence.
  • the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
  • FIG. 1 Sequence alignment of reference and codon optimized SIVmac239 virion infectivity factor (Vif).
  • A Nucleotide alignment of Vif coding DNA sequence from AY588946 (positions 4436-5080) (SEQ ID NO: l) and a codon optimized SIVmac239 Vif coding DNA sequence (SEQ ID NO:2). Identical residues are shaded grey; these two sequences are 76% identical.
  • B Amino acid alignment of the translated Vif coding DNA sequence described above (SEQ ID NO:3). These two protein sequences are 100% identical.
  • FIG 2. SIVmac239 Vif efficiently degrades human APOBEC3B (huA3B).
  • FIG 3. Degradation of huA3B by diverse SIV Vif proteins. Representative immunoblot demonstrating the abilities of Vif from SIVsmCFU212, SIVsmPG, SIVsmPBj, SIVsmE041, SIVstm, SIVmacl42, SIVmfal86, SIVmne07, SIVsmE543, and SIVagmTAN lentiviruses to degrade huA3B, in comparison to HIV- I HIB and SIVmac239 Vif proteins. With the exception of Vif from SIVagmTAN, the ability to mediate degradation of huA3B appears conserved in SIV Vif proteins. The lysates were blotted for myc to detect Vif, HA to detect huA3B, and tubulin (TUB) as a loading control.
  • Vif SIVsmCFU212, SIVsmPG, SIVsmPBj, SIVsmE041, SIVstm,
  • FIG 4. A3B restricts HIV-1 and is counteracted by SIVmac239 Vif.
  • 293T cells were transfected with Vif-deficient HIV- I HIB and with either empty expression constructs or the indicated A3 expression constructs (huA3B-HA, rhA3B-HA, or huA3G-HA) and Vif expression constructs (HIV-lniB Vif, or SIVmac239 Vif). After 48 hours, virus-containing supernatants from the 293 T cells were purified and used to infect CEM-GFP reporter cells.
  • Representative immunoblots for each infection condition vector control, HIV-l niB Vif, or SIVmac239 Vif; and vector control, huA3B-HA expression construct, rhA3B-HA expression construct, or huA3G-HA expression construct
  • 293T producer cells lysates were blotted for HA to detect A3, for Myc to detect Vif, and for Tubulin (TUB) as a loading control.
  • Purified viral particles were blotted for HA to detect A3 and for p24 (Gag) as a loading control.
  • FIG 5. SIVmac239 Vif-mediated degradation of huA3B is analogous to HIV- ⁇ ⁇ Vif- mediated degradation of huA3G.
  • A 293T cells were transfected with the indicated constructs, allowed to incubate for 32 hours, then treated with 5 uM of the proteasomal inhibitor MG132 or vehicle (40% acetonitrile in water) for 16 hours, processed into soluble lysates, and subjected to immunoblotting. These representative immunoblots demonstrate that MG132 treatment prevents Vif-mediated degradation of A3 proteins.
  • B Amino acid alignment of the ElonginC (ELOC)- binding SLQ region of the HIV-1 and SIV Vif proteins used in this study. conserveed residues are shaded, with underlining identifying more conserved positions. The residue positions included in the alignment are indicated. The SLQ tri-residue motif is underlined.
  • C Representative
  • FIG. SIVmac239 Vif-mediated rescue of cells from huA3B-mediated DNA damage and cytotoxicity.
  • B Representative assay for T-REx 293 cells expressing A3B-GFP with doxycycline induction and either stably expressing vector (circles), HIV-I HIB Vif (diamonds), or SIVmac239 Vif (triangles). Relative vi
  • immunoblot for cells as described in part (C) at each doxycycline concentration shows induction of GFP in the presence of the indicated Vif constructs.
  • the lysates were blotted for GFP and Hsp90 as a loading control.
  • FIG 7. SIVmac239 Vif can degrade endogenous huA3B in cancer cells. Immunoblot of
  • HCC1569 breast cancer cell line
  • JSQ3 head and neck cancer cell line
  • OVCAR-5 ovarian cancer cell line
  • the lysates were blotted for endogenous A3B using rabbit polyclonal anti-A3B (10-87-13), myc to detect Vif, and tubulin (TUB) as a loading control.
  • DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS APOBEC3B can be a source of mutations in breast and several other types of cancer.
  • HIV-1 protein virion infectivity factor (Vif) counteracts the effect of APOBEC3 protein restriction at least in part by targeting the APOBEC3 proteins for ubiquitin ligase-mediated proteasomal degradation.
  • APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif.
  • SIV simian immunodeficiency virus
  • This disclosure describes the use of simian immunodeficiency virus (SIV) Vif, including modifications, derivatives and conjugates thereof, for use in neutralizing and/or degrading APOBEC3B.
  • SIV simian immunodeficiency virus
  • This disclosure also describes vectors including nucleic acids encoding SIV Vif.
  • SIV Vif as a therapeutic agent, for example, to treat cancers, precancerous conditions, growth of tumors, or inhibit genetic evolution of tumors.
  • APOBEC3B unlike other APOBEC3 family members does not restrict HIV-1 and is not targeted by HIV-1 Vif.
  • a different lentiviral Vif protein could degrade APOBEC3B
  • Vif from SIVmac239 was identified as a potent APOBEC3B antagonist.
  • the ability of Vif to rescue cells overexpressing APOBEC3B from cell death was tested, and SIVmac239 Vif was found to be able to effectively counteract APOBEC3B-mediated cytotoxicity.
  • the APOBEC3B degradation potential of SIVmac239 Vif may be an effective strategy for efficiently neutralizing the cancer genomic DNA deaminase APOBEC3B.
  • the present disclosure describes a simian immunodeficiency virus (SIV) Vif that may be used to inhibit, neutralize, degrade and/or modulate the functional activity of
  • SIV simian immunodeficiency virus
  • a modulation in a functional activity can be quantitatively measured and described as a percentage of the functional activity of a comparable control.
  • APOBEC3B includes a modulation that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%), at least 30%>, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 110%, at least 125%, at least 150%, at least 200%, or at least 250%) of the activity of a suitable control.
  • the stimulation of a functional activity of an APOBEC3B polypeptide can be quantitatively measured and described as a percentage of the functional activity of a comparable control.
  • Stimulation of a functional activity of an APOBEC3B polypeptide includes a stimulation that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 110%, at least 125%, at least 150%, at least 200%, or at least 250% greater than the activity of a suitable control.
  • inhibition of a functional activity of an APOBEC3B polypeptide can be quantitatively measured and described as a percentage of the functional activity of a comparable control.
  • Inhibition of a functional activity of an APOBEC3B polypeptide includes an inhibition that is no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, no more than 50%, no more than 55%, no more than 60%, no more than 65%, no more than 70%, no more than 75%, no more than 80%, no more than 85%, no more than 90%, no more than 95%, no more than 99%, or no more than 100% of the activity of a suitable control.
  • the simian immunodeficiency virus (SIV) Vif may be, for example, SIVmac239 Vif, SIVsmCFU212 Vif, SIVsmPG Vif, SIVsmPBj Vif, SIVsmE041 Vif, SIVstm Vif, SIVmacl42 Vif, SIVmfal86 Vif, SIVmne07 Vif, and/or SIVsmE543 Vif, etc.
  • the SIV Vif may be SIVmac239 Vif.
  • the SIV Vif may include the polypeptide sequence SEQ ID NO:3.
  • the disclosure provides polynucleotides that encode any of the SIV Vif polypeptides or portions of the SIV Vif polypeptides described herein, and the polynucleotide sequences that can encode such polypeptide sequences. Given the amino acid sequence of any one of the SIV Vif polypeptides described herein, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods.
  • the coding sequence of SIV Vif may be modified to increase expression in a mammal including, for example, a mouse, a rat, a human, etc.
  • the coding sequence of SIV Vif may be codon optimized. In some embodiments, the coding sequence of SIV Vif may be codon optimized using OPTIMUMGE E (GenScript USA, Inc., Piscataway, NJ). The optimization for expression including, for example, codon optimization, may take into account factors such as, for example, codon adaptability, mRNA structure, various c/5-elements in transcription and translation, etc. In some embodiments, the coding sequence of SIV Vif may include SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, the polypeptide sequence of SIV Vif includes a conserved SLQ tri- residue peptide motif and/or a conserved TLQ tri-residue peptide motif.
  • the SLQ tri-residue peptide motif and/or a conserved TLQ tri-residue peptide motif may be involved in binding to an E3 ubiquitin ligase or E3 ubiquitin ligase complex.
  • the SLQ tri-residue motif and/or TLQ tri-residue motif of SIV Vif may be involved in binding to ElonginC (ELOC).
  • a polynucleotide that encodes an SIV Vif polypeptide or portions of an SIV Vif polypeptide may be introduced into a cell.
  • the polynucleotide or portions of the polynucleotide may be introduced into the cell by transfection, transduction, and/or infection.
  • the polynucleotide may be in a vector, a small particle (e.g., a virus-like particle), a viral particle, etc.
  • a vector containing a polynucleotide encoding SIV Vif may be used to transfect, transduce, and/or infect cells, including, for example, cancerous or pre-cancerous cells.
  • cells into which the polynucleotide is introduced are human cells including, for example, human tumor cells.
  • the expression of the SIV Vif in the transfected, transduced, or infected cell may result in neutralizing and/or degrading APOBEC3B in the transfected, transduced, or infected cell.
  • expression of the SIV Vif in the transfected, transduced, or infected cell may result in abated mutagenesis of the cell's DNA.
  • Exemplary vectors that may be designed to contain a polynucleotide encoding an SIV Vif polypeptide or a portion of an SIV Vif polypeptide include, for example, plasmids, cosmids, bacteriophages, viral vectors, etc.
  • the vector may contain a selectable marker for propagation in a host.
  • the vector may be a gene therapy -type viral vector including, for example, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a pox virus, an alphavirus, a herpes virus, a measles virus, an influenza virus, etc.
  • a vector that contains a polynucleotide encoding an SIV Vif may include one or more expression regulatory elements, such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal and/or an enhancer. Each of these elements may be operably linked.
  • the vector may further contain a site for transcription initiation, a site for transcription, a site for termination and/or, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of a mature transcript expressed by the constructs may include a translation initiation at the beginning and/or a termination codon appropriately positioned at the end of the polypeptide to be translated.
  • a viral particle including a polynucleotide that encodes an SIV Vif polypeptide or portions of an SIV Vif polypeptide may include, for example, a viral particle derived from an enveloped- virus, for example a retroviruses, including, for example, a rous sarcoma virus, a human and/or bovine T-cell leukemia virus (HTLV and BLV), a lentivirus such as human and simian
  • immunodeficiency viruses (HIV and SIV), Mason-Pfizer monkey virus; a foamy virus; a herpes virus, including for example, HSV, varicella-zoster, vaccinia; a Pox virus; an orthomyxovirus including, for example, influenza; a paramyxovirus, including, for example, a parainfluenza virus, a respiratory syncytial virus, a Sendai virus, a mumps virus, a measles virus; a corona virus, a flavivirus; an alphavirus; a rhabdovirus including, for example, a vesicular stomatitis virus, a rabies virus; a vaccinia virus; a bunyavirus, and/or an RNA virus, including, for example, a Rhabdovirus VSV (vesicular stomatitis virus).
  • a paramyxovirus including, for example, a parainfluenza virus,
  • This disclosure further provides a method that includes administering a polynucleotide encoding an SIV Vif as an active ingredient in an anticancer composition.
  • the anticancer composition can include a
  • pharmaceutically acceptable carrier can be prepared into various dosage forms including tablets, troches, capsules, elixirs, suspensions, creams, syrups, wafers, injections, etc.
  • any form of solvent, dispersing media, antibacterial agent, antifungal agent, isotonic, absorption retardant, or any combination thereof may be used as a carrier.
  • solvent, dispersing media, antibacterial agent, antifungal agent, isotonic, absorption retardant, or any combination thereof may be used as a carrier.
  • composition can be formulated into a suitable pharmaceutical preparation.
  • a vector, a viral particle, or a small particle containing a polynucleotide encoding an SIV Vif may be administered via an oral route or a non-oral route such as, for example, an intramuscular, intravenous, peritoneal, subcutaneous, or intradermal route.
  • An injection may be, for example, an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, instillation, or an intratumoral injection.
  • the composition may be administered in a single dose or in multiple doses.
  • the composition may be administered either locally— e.g., to the location of a tumor— or systemically.
  • the anticancer composition may be administered in a pharmaceutically effective amount.
  • the pharmaceutically effective amount may depend, at least in part, on the route of administration, frequency of administration, the kind of the cancer being treated, the severity of the cancer being treated, the age of the patient, the gender of the patient, the condition of the patient, and/or other factors well-known in the pharmaceutical art.
  • the pharmaceutically effective amount may be administered once or in multiple doses.
  • the disclosure further provides a therapeutic method of treating a subject suffering from a cancer or a precancerous condition.
  • the method includes administering a vector including a polynucleotide encoding a SIV Vif to the subject.
  • Therapeutic treatment is initiated after the development of cancer or a precancerous condition.
  • Administering a vector including a polynucleotide encoding a SIV Vif may be particularly advantageous for treating a cancer or a precancerous condition wherein the cancerous or precancerous cells are known to express APOBEC3B.
  • Expression of APOBEC3B may be determined using various methods including, for example, quantitative PCR and/or detection with anti-A3B antibodies (including those described in U.S. Provisional Patent Application No. 62/186, 109, filed June 29, 2015).
  • the therapeutic method can be used to treat a variety of cancerous or precancerous conditions, including tumors or dysplasia.
  • a tumor can be a solid tumor, such as a carcinoma, a sarcoma, or a lymphoma, and can be present, for example, in the bone, brain, breast, cervix, larynx, lung, pancreas, prostate, skin, spine, stomach, bladder, uterus, etc.
  • the cancer treated can also be a blood cancer, such as leukemia or lymphoma.
  • the dysplasia can be an epithelia dysplasia.
  • the tumor can made up of tumor cells, including lymphoid and myeloid cancers; multiple myeloma; cancers of the bone, breast, prostate, stomach, colon, pancreas, or thyroid; melanoma; head and neck squamous cell carcinoma; ovarian carcinoma; or cervical carcinoma.
  • tumor cells including lymphoid and myeloid cancers; multiple myeloma; cancers of the bone, breast, prostate, stomach, colon, pancreas, or thyroid; melanoma; head and neck squamous cell carcinoma; ovarian carcinoma; or cervical carcinoma.
  • a precancerous condition may include infection with a virus that increases APOBEC3 expression (Vieira et al., 2014, mBio. 5(6): e02234-14). In some
  • a precancerous condition may include infection with human papilloma virus (HPV).
  • HPV human papilloma virus
  • a precancerous condition may include a cervical abnormality.
  • a precancerous cell may include a HPV-infected cervical cell.
  • a vector including a polynucleotide encoding a SIV Vif can occur before, during, and/or after other treatments. Such combination therapy can involve the administration of vector including a polynucleotide encoding a SIV Vif before, during and/or after the use of other anti-cancer agents, for example, chemotherapeutic agents or radiation or both. It is expected that a vector including a polynucleotide encoding a SIV Vif may potentiate the effects of cytokines, chemotherapeutic agents, or gamma radiation.
  • the administration of vector including a polynucleotide encoding a SIV Vif can be separated in time from the administration of other anticancer agents by hours, days, or even weeks. Additionally or alternatively, the administration of a vector including a polynucleotide encoding a SIV Vif can be combined with other biologically active agents or modalities such as, but not limited to, an antineoplastic agent, and non-drug therapies, such as, but not limited to, surgery.
  • APOBEC3 expression constructs The APOBEC3 proteins huA3B (GenBank Accession No. (gb) NM_004900), huA3G (gb NM021822), and rhA3B (gb JF714485, but with the asparagine at amino acid residue 316 corrected to aspartic acid (McDougle et al. Virology 2013, 441 :31-39) were expressed with carboxy -terminal HA tag in the pcDNA3.1(+) vector (Invitrogen Corp., Carlsbad, CA). RhA3B cDNA was generously provided by Dr.
  • huA3B was expressed with a carboxy-terminal eGFP tag in the pcDNA5TO vector (Clontech Laboratories, Inc., Mountain View, CA).
  • Vif expression constructs The coding DNA sequences of lentiviral Vif proteins from HIV- I IIIB (protein sequence matches gb EU541617), SIVmac239 (protein sequence matches gb
  • BIV BIMI27 protein sequence matches gb M32690
  • MVV 1514 protein sequence matches gb M60610
  • FIV N SCU protein sequence matches gb m25381
  • SIVmac239 Vif has a cDNA sequence that does not exist in nature (FIG. 1 A), but the encoded protein matches gb AY588946 (FIG. IB).
  • the codon optimized coding DNA sequences of lentiviral Vif proteins from HIV- IHIB, SIVmac239, BIVBIMI27, MVV1514 , and FIVNSCU were used in this Example and FIGS. 2-7.
  • Vif expression constructs from SIVsmCFU212 gb JX860407
  • SIVmac239 Vif were subcloned into the pLenti4-Hygro-TO backbone, transduced into OVCAR-5 cells, and a stably expressing pool was selected with hygromycin.
  • HIV constructs The Vif proficient and Vif deficient (X26X27) HIV-I B A200C proviral expression constructs (gb EU541617) have been reported previously (Hache et al., 2008, Curr. Biol. 18:819-824).
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • P/S penicillin-streptomycin
  • CEM-GFP cells obtained from the NIH AIDS Reagent Program (Gervaix et al., 1997, Proc Natl Acad Sci USA 94:4653-4658), HCC1569 cells (ATCC CRL-2330, ATCC, Manassas, VA), and OVCAR-5 cells (obtained from the Mayo Clinic ovarian cell line repository; Monks et al., 1991, J. Natl. Cancer Inst. 83 :757-766) were maintained in RPMI medium with 10% FBS and 0.5% P/S.
  • tubulin was detected with monoclonal mouse anti-a- Tubulin (Covance Inc., Princeton, NJ)
  • HIV-1 Gag was detected with monoclonal mouse anti-HIV-1 p24 (NIH AIDS Reagent Program) (Chesebro et al., 1992, J. Virol. 66:6547- 6554)
  • A3-GFP was detected with monoclonal mouse anti-GFP (Clontech Laboratories, Inc., Mountain View, CA)
  • Hsp90 was detected with mouse anti-HSP90 (BD Biosciences, Franklin Lakes, NJ).
  • A3B was detected with rabbit polyclonal anti-A3B (10-87-13) (described in U.S.
  • Vif degradation 293T cells were transfected in triplicate with pVR1020-Vif-myc or empty vector, at levels normalized by immunoblot, and pcDNA3.1 A3-HA, or empty vector, as indicated, using PEI (polyethyleneimine) (Polysciences, Inc., Warrington, PA). After 48 hours, the cells were harvested for immunoblot analysis. To inhibit proteasomal degradation, MG132 (American Peptide, Sunnyvale, CA) was added at 5 ⁇ , 16 hours before harvesting the cells.
  • PEI polyethyleneimine
  • HIV-1 single cycle infection with replication-proficient virus The single cycle infectivity assays were performed as previously reported (Hultquist et al., 2011, J. Virol. 85: 11220-11234) by transfecting 293T cells (TransIT-LTl Transfection Reagent, Minis Bio LLC, Madison, WI) in triplicate with 1 ⁇ g of a Vif deficient HIV-1 proviral expression construct along with 25 ng of A3- HA expression construct or empty vector, and 25 ng of myc-tagged Vif expression construct or empty vector. After 48 hours, purified virus-containing supernatants were used to infect the CEM- GFP HIV-1 reporter cells, and cell and viral particle lysates were prepared for immunoblotting. Infectivity was normalized to the vector control.
  • Flow cytometry HIV-infected CEM-GFP cells were prepared for flow cytometry by fixation in 4% paraformaldehyde. GFP fluorescence was measured on a BD FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ). All data were analyzed using FlowJo flow cytometry analysis software (version 8.8.7). GFP fluorescence was quantified from the gated live cell population.
  • T-REx 293 cells (Invitrogen Corp., Carlsbad, CA), which stably express the tetracycline repressor, were transfected with pcDNA5TO-A3B-eGFP and pcDNA5TO-eGFP constructs using TransIT-LTl (Minis Bio LLC, Madison, WI). Stable clones were selected with hygromycin and blasticidin.
  • T-REx 293 huA3B and T-REx 293 GFP stable clones were further engineered to stably express HIV-I HIB Vif-myc, SIVmac239 Vif-myc or vector by transfection of pcDNA3.1 expression constructs and selection with G418.
  • SIVmac239 Vif potently counteracts human A3B.
  • HIV- I HIB Vif does not mediate degradation of huA3B.
  • the ability of a panel of Vif constructs derived from HIV- I B , SIVmac239, BIV, MLV and FIV to mediate degradation of huA3B was tested. These Vif constructs were transfected into 293T cells at equivalent levels, based on immunoblot, along with a constant amount of huA3B or vector control (FIG. 1).
  • HIV-I HIB Vif was able to mediate degradation of huA3B at the highest levels of expression, but did not have any effect on huA3B at the lower levels.
  • SIVmac239 Vif was able to mediate degradation of huA3B at all tested expression levels, with the lowest level of SIVmac239 Vif mediating a similar level of huA3B degradation as the highest level of HIV- I HIB Vif, and the highest level of SIVmac239 Vif rendering huA3B barely detectable by immunoblot (FIG. IB).
  • huA3B cotransfected with BIV Vif showed moderately lower levels of expression, regardless of the amount of BIV Vif co-transfected.
  • FIV Vif and MVV Vif did not have any effect on huA3B, regardless of expression level.
  • a panel of SIV Vif expression constructs including lentiviruses that infect sooty
  • SIVmac239 Vif mediates degradation of huA3B in a manner analogous to HIV- I HIB Vif mediated degradation of huA3G.
  • SIVmac239 Vif mediates degradation of huA3B in a conserved manner to HIV- I HIB Vif degradation of huA3G - an interaction that has been extensively studied - Vif mediated relief of HIV restriction in a single cycle assay was tested.
  • rhA3B susceptibility to SIVmac239 Vif was examined, as rhA3B is the cis-species target of SIVmac239. Vif.
  • huA3B, rhA3B, huA3G, and vector control constructs were transfected into 293T cells with Vif-deficient full-length molecular clone HIV- I IUB .
  • HIV- I HIB Vif and SIVmac239 Vif were also transfected into the cells on separate expression vectors.
  • huA3G restricted the viral infectivity in the absence of any Vif protein, but not when HIV-I IIIB Vif was present (FIG. 4A).
  • the ability of huA3G to restrict HIV replication was also counteracted by SIVmac39 Vif (FIG. 4A).
  • huA3G was degraded in the presence of both HIV- I IIIB and SIVmac239 Vif (FIG. 4B).
  • huA3B restricted HIV replication both in the absence of Vif protein, and in the presence of HIV-I HIB Vif.
  • SIVmac239 Vif alone was able to relieve huA3B restriction of HIV replication (FIG. 4 A), and only SIVmac239 Vif mediated degradation of huA3B (FIG. 4B).
  • the rhA3B protein showed a similar restriction profile to huA3B.
  • rhA3B was restrictive in the absence of Vif, and in the presence of HIV- I HIB Vif.
  • SIVmac239 Vif moderately restored viral infectivity in the presence of rhA3B (FIG. 4 A), and had a small effect on rhA3B degradation (FIG. 4B).
  • huA3G was degraded in the presence of HIV-1 HIB Vif and SIVmac239 Vif in the absence of MG132, but in the presence of proteasomal inhibition, huA3G degradation was inhibited (FIG. 5A).
  • rhA3B was degraded in the presence of SIVmac239 Vif, while HIV-I HIB Vif was not observed to mediate degradation of rhA3B. Inhibition of the
  • proteasome with MG132 decreased SIVmac239 Vif mediated degradation of rhA3B (FIG. 5A).
  • SIVmac239 Vif are only 30% identical at the amino acid level, but the SLQ motif is conserved. In fact, this motif is conserved in all the SIV Vif strains used in this study (FIG. 5B).
  • Cells were transfected with APOBEC3 and Vif constructs as shown in FIG. 5C.
  • Mutation of the SLQ region to AAA in HIV- I IIIB Vif abrogated its ability to mediate degradation of huA3G.
  • mutation of the SLQ region to AAA in SIVmac239 Vif also abolished degradation of huA3G (FIG. 5C).
  • SIVmac239 Vif rescues cells from huA3B mediated cytotoxicity When overexpressed, huA3B is toxic and kills cells in a dose-dependent manner (Burns et al., 2013, Nature 494:366- 370).
  • huA3B-GFP or GFP alone was stably expressed under the control of a doxycycline-inducible promoter in T-REx 293 cells, allowing for titratable expression of the protein.
  • Vector control, HIV- I IIIB Vif, and SIVmac239 Vif were stably transfected into the inducible huA3B and GFP cells, and their expression was confirmed by immunoblot (FIG. 6A, inset, and FIG. 6B, inset). These cells were plated for clones in increasing concentrations of doxycycline to assess viability in the presence of Vif and the inducible presence of huA3B.
  • stable expression of HIV-I HIB Vif decreased detected huA3B protein levels compared to no Vif (FIG. 6B).
  • SIVmac239 Vif expression indicating that in the absence of huA3B, these Vif proteins have no effect on viability.
  • SIVmac239 Vif degrades endogenously expressed huA3B.
  • HCC1569 cells a human breast cancer cell line
  • JSQ3 a human head and neck cancer cell line
  • OVCAR5 a human ovarian cancer cell line.
  • HIV- I HIB and SIVmac239 Vif were stably expressed and cell lysates were immunoblotted for huA3B.
  • Cells expressing vector and HIV-I IIIB Vif exhibited comparable levels of endogenous huA3B (FIG. 7).
  • all three cell lines engineered to express SIVmac239 Vif showed lower levels of huA3B, indicating that
  • SIVmac239 Vif is capable of mediating degradation of endogenous huA3B in cancer cells (FIG. 7).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Dans un aspect, un procédé consiste à fournir des cellules tumorales, fournir un polynucléotide codant au moins une partie d'un gène de virus d'immunodéficience simienne (SIV) Vif, et introduire le polynucléotide dans les cellules tumorales. Dans un autre aspect, un procédé consiste à introduire, dans une cellule, un polynucléotide codant un virus d'immunodéficience simienne (SIV) Vif, l'expression du Vif SIV dans la cellule entraînant la modulation de l'activité fonctionnelle d'APOBEC3B dans la cellule. Dans l'un ou l'autre aspect, la protéine Vif SIV peut comprendre SIVmac239 Vif, SIVsmCFU212 Vif, SIVsmPG Vif, SIVsmPBj Vif, SIVsmE041 Vif, SIVstm Vif, SIVmacl42 Vif, SIVmfal86 Vif, SIVmne07 Vif ou SIVsmE543 Vif. Dans certains de ces modes de réalisation, le polynucléotide est optimisé par codons pour l'expression dans une cellule humaine.
PCT/US2016/039973 2015-06-29 2016-06-29 Dégradation ou neutralisation d'apobec3b par le virus d'immunodéficience simienne vif WO2017004130A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562186256P 2015-06-29 2015-06-29
US62/186,256 2015-06-29

Publications (1)

Publication Number Publication Date
WO2017004130A1 true WO2017004130A1 (fr) 2017-01-05

Family

ID=57609419

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/039973 WO2017004130A1 (fr) 2015-06-29 2016-06-29 Dégradation ou neutralisation d'apobec3b par le virus d'immunodéficience simienne vif

Country Status (1)

Country Link
WO (1) WO2017004130A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020190609A1 (fr) * 2019-03-15 2020-09-24 Mayo Foundation For Medical Education And Research Virus oncolytiques et méthodes d'utilisation des virus oncolytiques

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851813A (en) * 1990-07-12 1998-12-22 President And Fellows Of Harvard College Primate lentivirus antigenic compositions
US20080226675A1 (en) * 2007-03-17 2008-09-18 Hongzhan Xu Recombinant vector and use in gene therapy
US20100285061A1 (en) * 2007-01-12 2010-11-11 The Government of the United States, as represented by The Secretary of The Department ... Dna vaccination protocols
US20110212530A1 (en) * 2005-06-01 2011-09-01 California Institute Of Technology Method of targeted gene delivery using viral vectors
WO2012168480A1 (fr) * 2011-06-10 2012-12-13 Institut Curie Agents et procédés pour la production d'adjuvants non infectieux dérivés de capsides de vih

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851813A (en) * 1990-07-12 1998-12-22 President And Fellows Of Harvard College Primate lentivirus antigenic compositions
US20110212530A1 (en) * 2005-06-01 2011-09-01 California Institute Of Technology Method of targeted gene delivery using viral vectors
US20100285061A1 (en) * 2007-01-12 2010-11-11 The Government of the United States, as represented by The Secretary of The Department ... Dna vaccination protocols
US20080226675A1 (en) * 2007-03-17 2008-09-18 Hongzhan Xu Recombinant vector and use in gene therapy
WO2012168480A1 (fr) * 2011-06-10 2012-12-13 Institut Curie Agents et procédés pour la production d'adjuvants non infectieux dérivés de capsides de vih

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, W ET AL.: "Cytidine Deaminase APOBEC3B Interacts With Heterogeneous Nuclear Ribonucleoprotein K And Suppresses Hepatitis B Virus Expression.", CELLULAR MICROBIOLOGY., vol. 10, no. 1, 1 August 2007 (2007-08-01), pages 112 - 121, XP055344519 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020190609A1 (fr) * 2019-03-15 2020-09-24 Mayo Foundation For Medical Education And Research Virus oncolytiques et méthodes d'utilisation des virus oncolytiques

Similar Documents

Publication Publication Date Title
Carnes et al. HIV-1 engages a dynein-dynactin-BICD2 complex for infection and transport to the nucleus
CA2456169C (fr) Procedes et compositions relatifs a des systemes production ameliores de vecteurs lentiviraux
JP7350485B2 (ja) ウイルスベクター効率を改善するための組成物及び方法
Trunschke et al. The L–VP35 and L–L interaction domains reside in the amino terminus of the Ebola virus L protein and are potential targets for antivirals
ES2911448T3 (es) Vectores combinados y métodos para el tratamiento del cáncer
Dietrich et al. Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection
Yin et al. Equine tetherin blocks retrovirus release and its activity is antagonized by equine infectious anemia virus envelope protein
Tang et al. Equine viperin restricts equine infectious anemia virus replication by inhibiting the production and/or release of viral Gag, Env, and receptor via distortion of the endoplasmic reticulum
US20120027725A1 (en) Safe lentiviral vectors for targeted delivery of multiple therapeutic molecules to treat liver cancer
Gonzalez-Enriquez et al. SERINC as a restriction factor to inhibit viral infectivity and the interaction with HIV
Land et al. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif
Nepveu-Traversy et al. The conserved sumoylation consensus site in TRIM5α modulates its immune activation functions
Zhang et al. Inhibitory effect of human TRIM5α on HIV-1 production
Lucas et al. Pseudotyping incompatibility between HIV-1 and gibbon ape leukemia virus Env is modulated by Vpu
JP3954151B2 (ja) フォーミーウイルスエンベロープタンパク質の発現
WO2017004130A1 (fr) Dégradation ou neutralisation d'apobec3b par le virus d'immunodéficience simienne vif
CA3177006A1 (fr) Compositions et procedes pour le traitement de la fibrose kystique
US20140010839A1 (en) Method of screening antiretroviral compounds and vaccine
Nepveu-Traversy et al. A putative SUMO interacting motif in the B30. 2/SPRY domain of rhesus macaque TRIM5α important for NF-κB/AP-1 signaling and HIV-1 restriction
Marcello et al. Inducible expression of herpes simplex virus thymidine kinase from a bicistronic HIV1 vector
Garg et al. Conditional cytotoxic anti-HIV gene therapy for selectable cell modification
US20090202488A1 (en) Use Of The Foamy Virus Bet Protein For Inactivating APOBEC
WO2016018921A1 (fr) Compositions et procédés pour inhiber le bourgeonnement du virus du syndrome immunodéficitaire acquis
EP1832604A1 (fr) Utilisation de IEX-1 pour le traitement des gliomes
WO2012118092A1 (fr) Protéine hybride

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16818642

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16818642

Country of ref document: EP

Kind code of ref document: A1