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WO2002072756A2 - Lambeau d'adn central du virus de l'immunodeficience feline - Google Patents

Lambeau d'adn central du virus de l'immunodeficience feline Download PDF

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WO2002072756A2
WO2002072756A2 PCT/US2002/006818 US0206818W WO02072756A2 WO 2002072756 A2 WO2002072756 A2 WO 2002072756A2 US 0206818 W US0206818 W US 0206818W WO 02072756 A2 WO02072756 A2 WO 02072756A2
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sequence
vector
nucleic acid
fiv
seq
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WO2002072756A3 (fr
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Eric M. Poeschla
Todd A. Whitwam
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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    • 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
    • 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

  • the invention relates to methods and materials involved in making and using feline immunodeficiency virus (FIN) lentiviral vectors.
  • FIN feline immunodeficiency virus
  • Feline immunodeficiency virus is a lentivirus that infects non-dividing cells, causes progressive CD4+ T cell depletion in domestic cats but not in humans, and has been used as a substrate for lentiviral vectors.
  • the invention involves lentiviral vectors.
  • the invention provides for lentiviral vectors comprising a nucleotide sequence having at least 90% identity to the FIV central polypurine tract (cPPT) of any FIN strain or clone provided herein.
  • lentiviral vectors can also include a central termination sequence (CTS) that is capable of functioning in conjunction with a cPPT to generate a central D ⁇ A flap required for nuclear import of a pre-integration complex through an intact nuclear membrane.
  • CTS central termination sequence
  • Figure 1 is a diagram depicting the location of the cPPT, the CTS, and the intervening sequence that comprise the central flap in FIN.
  • Figure 2A and 2B are bar graphs of infectious titers from either dividing (2 A) or non-dividing (2B) cells.
  • CT25a is the control vector lacking an FIN cPPT
  • CT26 is the vector containing an FIV cPPT
  • CT26r is the vector containing an antisense FIV cPPT.
  • the invention provides methods and materials related to making and using lentiviral vectors. Specifically, the invention provides vectors comprising nucleic acids and host cells comprising lentiviral vectors or particles.
  • central polypurine tract or "cPPT” as used herein refers to a nucleic acid sequence that comprises a plurality of contiguous purines (i.e., A or G) that can be interrupted by one or more pyrimidines (i.e., T, C, or U).
  • a cPPT is, without limitation, at least about 16 nucleotides in length.
  • a group of related cPPTs can contain a conserved pyrimidine.
  • the related FIV sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7 contain a conserved thymidine located at position 4970 of the FIV genome (for FIV numbering conventions see Talbott et al., Proc. Nat. Acad. Sci. USA 86:5743-7 (1989)), or, in other words, -2 to the G nucleotide that defines the 5' terminus of the downstream (D+) strand.
  • central gap refers to a single-stranded discontinuity located in the approximate center of an FIV genome.
  • LMW low molecular weight
  • gap nucleotide refers to a nucleotide that defines the 3' boundary of a central gap, e.g., the 5' terminus of the D+ strand.
  • the underlined "G” in the FIV sequence 5'- AAAAGAAGAGGTAGGA-3' is the gap nucleotide that identifies the 5' terminus of the D+ strand for the FIV strain comprising SEQ ID NO: 1.
  • central termination sequence refers to a site in a viral genome where upstream (U+) strand synthesis is terminated.
  • CTS central termination sequence
  • the invention also provides intervening nucleic acid sequences.
  • intervening sequence refers to a nucleic acid sequence flanked by a cPPT and a CTS.
  • the cPPT and CTS can each be either 5 ' or 3' of the intervening sequence.
  • the cPPT can be 5' of the intervening sequence, with the CTS being 3' of the intervening sequence.
  • An intervening sequence can vary in length, e.g., can be 8, 9, 10, 15, 20, 50, 100, 200, or 500 nucleotides in length.
  • insert nucleic acid refers to a nucleic acid sequence of interest that has been, or is to be, inserted into a transfer vector.
  • an insert nucleic acid is distinct from a cPPT, a CTS, or an intervening sequence as they relate to the invention described herein.
  • a transfer vector can contain an insert nucleic acid encoding a marker polypeptide, e.g., green fluorescent protein (GFP), or any other polypeptide.
  • GFP green fluorescent protein
  • an insert nucleic acid can be a non-coding nucleic acid, e.g., an antisense nucleic acid.
  • transfer vector refers to a nucleic acid vector that has been adapted to receive an insert nucleic acid.
  • a transfer vector within the scope of the invention includes vectors containing or not containing an insert nucleic acid.
  • nucleic acid encompasses RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA.
  • the nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.
  • isolated as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally-occurring genome of the organism from which it is derived.
  • an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote.
  • an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.
  • isolated as used herein with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occu ⁇ ing nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally- occurring genome.
  • non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid.
  • Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques.
  • Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote.
  • a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.
  • nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.
  • exogenous as used herein with reference to nucleic acid and a particular cell refers to any nucleic acid that does not originate from that particular cell as found in nature. Thus, all non-naturally-occurring nucleic acid is considered to be exogenous to a cell once introduced into the cell.
  • non- naturally-occurring nucleic acid can contain nucleic acid sequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature.
  • a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a cell once introduced into the cell, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature.
  • any vector, autonomously replicating plasmid, or virus that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid.
  • genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature.
  • any nucleic acid containing a promoter sequence and polypeptide-encoding sequence e.g., cD A or genomic DNA
  • cD A or genomic DNA in an arrangement not found in nature is non-naturally- occurring nucleic acid.
  • Nucleic acid that is naturally-occurring can be exogenous to a particular cell.
  • an entire chromosome isolated from a cell of person X is an exogenous nucleic acid with respect to a cell of person Y once that chromosome is introduced into Y's cell.
  • the invention provides isolated nucleic acids that contain a nucleic acid sequence at least about 90% identical to nucleotides 5-20 of the sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7.
  • the percent identity between two nucleic acid sequences is determined as follows.
  • a nucleic acid sequence is compared to nucleotides 5-20 of a sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14.
  • This stand-alone version of BLASTZ can be obtained from www.fr.com or www.ncbi.nlm.nih.gov.
  • Instractions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ.
  • B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C: ⁇ seql.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C: ⁇ seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C: ⁇ output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting.
  • the following command can be used to generate an output file containing a comparison between two sequences: C: ⁇ B12seq -i c: ⁇ seql .txt -j c: ⁇ seq2.txt — p blastn -o c: ⁇ output.txt -q -1 -r 2. If the two compared nucleic acid sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared nucleic acid sequences do not share homology, then the designated output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
  • percent identity value is rounded to the nearest tenth.
  • 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1
  • 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2.
  • the length value will always be an integer.
  • the invention also provides isolated nucleic acid molecules that are at least about 5 bases in length (e.g., at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 100, 250, 500, 750, 1000, 1500, 2000, 3000, 4000, or 5000 bases in length) and hybridize, under hybridization conditions, to the sense or antisense strand of a nucleic acid having a sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7.
  • the hybridization conditions can be moderately or highly stringent hybridization conditions.
  • moderately stringent hybridization conditions mean the hybridization is performed at about 42°C in a hybridization solution containing 25 mM KPO 4 (pH 7.4), 5X SSC, 5X Denhart's solution, 50 ⁇ g/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5x10 7 cpm/ ⁇ g), while the washes are performed at about 50°C with a wash solution containing 2X SSC and 0.1% sodium dodecyl sulfate.
  • Highly stringent hybridization conditions mean the hybridization is performed at about 42°C in a hybridization solution containing 25 mM KPO 4 (pH 7.4), 5X SSC, 5X Denhart's solution, 50 ⁇ g/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5xl0 7 cpm/ ⁇ g), while the washes are performed at about 65°C with a wash solution containing 0.2X SSC and 0.1% sodium dodecyl sulfate.
  • Isolated nucleic acid molecules within the scope of the invention can be obtained using any method including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques.
  • PCR can be used to obtain an isolated nucleic acid molecule containing a nucleic acid sequence sharing similarity to nucleotides 5-20 of the sequence set forth in SEQ ID NO: 1.
  • PCR refers to a procedure or technique in which target nucleic acid is amplified in a manner similar to that described in U.S. Patent No. 4,683,195, and subsequent modifications of the procedure described therein.
  • sequence information from the ends of the region of interest or beyond are used to design oligonucleotide primers that are identical or similar in sequence to opposite strands of a potential template to be amplified.
  • a nucleic acid sequence can be amplified from RNA or DNA.
  • a nucleic acid sequence can be isolated by PCR amplification from total cellular RNA, total genomic DNA, and cDNA as well as from bacteriophage sequences, plasmid sequences, viral sequences, and the like.
  • reverse transcriptase can be used to synthesize complimentary DNA strands.
  • Isolated nucleic acid molecules within the scope of the invention also can be obtained by mutagenesis.
  • an isolated nucleic acid containing nucleotides 5- 20 of the sequence set forth in SEQ ID NO: 1 can be mutated using common molecular cloning techniques (e.g., site-directed mutagenesis).
  • Possible mutations include, without limitation, deletions, insertions, and substitutions, as well as combinations of deletions, insertions, and substitutions.
  • nucleic acid and amino acid databases can be used to obtain an isolated nucleic acid molecule within the scope of the invention.
  • GenBank ® any amino acid sequence having some homology to a sequence set forth in SEQ ID NO: 8, 9, or 10 can be used as a query to search GenBank ® .
  • nucleic acid hybridization techniques can be used to obtain an isolated nucleic acid molecule within the scope of the invention. Briefly, any nucleic acid molecule having some homology to nucleotides 5-20 of the sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 can be used as a probe to identify a similar nucleic acid by hybridization under conditions of moderate to high stringency. Once identified, the nucleic acid molecule then can be purified, sequenced, and analyzed to determine whether it is within the scope of the invention as described herein.
  • Hybridization can be done by Southern or Northern analysis to identify a DNA or RNA sequence, respectively, which hybridizes to a probe.
  • the probe can be labeled with a biotin, digoxygenin, an enzyme, or a radioisotope such as 32 P.
  • the DNA or RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sa brook et al, (1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory, Plainview, NY.
  • a probe is at least about 16 nucleotides in length.
  • a probe corresponding to nucleotides 5-20 of the sequence set forth in SEQ ID NO: 1 can be used to identify an identical or similar nucleic acid, h addition, probes longer or shorter than 16 nucleotides can be used.
  • a host cell within the scope of the invention is any cell containing at least one isolated nucleic acid molecule described herein. Such cells can be prokaryotic and eukaryotic cells. It is noted that cells containing an isolated nucleic acid molecule within the scope of the invention are not required to express a polypeptide. In addition, the isolated nucleic acid molecule can be integrated into the genome of the cell or maintained in an episomal state. Thus, host cells can be stably or transiently transfected with a construct containing an isolated nucleic acid molecule of the invention.
  • Host cells within the scope of the invention can contain an exogenous vector that comprises a nucleotide sequence having 90% identity to nucleotides 5-20 of the sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7.
  • a somatic cell containing a vector comprising nucleotides 5-20 of the sequence set forth in SEQ ID NO: 1 is a host cell.
  • cultured cells e.g., Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK-21 cells, MDCK cells, or human vascular endothelial cells (HUVEC), containing an exogenous vector that comprises a nucleotide sequence having 90% identity to nucleotides 5-20 of the sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 can be host cells.
  • any methods can be used to introduce an isolated nucleic acid molecule into a cell in vivo or in vitro.
  • calcium phosphate precipitation, electroporation, heat shock, lipofection, micro injection, and viral-mediated nucleic acid transfer are common methods that can be used to introduce an isolated nucleic acid molecule into a cell.
  • naked DNA can be delivered directly to cells in vivo as describe elsewhere (U.S. Patent Number 5,580,859 and U.S. Patent Number 5,589,466 including continuations thereof).
  • isolated nucleic acid molecules can be introduced into cells by generating transgenic animals.
  • Transgenic animals can be aquatic animals (such as fish, sharks, dolphin, and the like), farm animals (such as pigs, goats, sheep, cows, horses, rabbits, and the like), rodents (such as rats, guinea pigs, and mice), non-human primates (such as baboon, monkeys, and chimpanzees), and domestic animals (such as dogs and cats).
  • Several techniques known in the art can be used to introduce isolated nucleic acid molecules into animals to produce the founder lines of transgenic animals. Such techniques include, without limitation, pronuclear microinjection (U.S. Patent No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad.
  • transgenic animals can be replicated using traditional breeding or animal cloning.
  • Any method can be used to identify cells containing an isolated nucleic acid molecule of the invention. Such methods include, without limitation, PCR and nucleic acid hybridization tecliniques such as Northern and Southern analysis. In some cases, immunohistochemistry and biochemical tecliniques can be used to determine if a cell contains a particular isolated nucleic acid molecule by detecting the expression of a polypeptide encoded by that particular nucleic acid molecule.
  • Example 1 Recovery of FIV pre-integration complexes by single-round infection
  • a single round infection assay that employs a VSV-G pseudotyped, env frame- shifted modification of CT5 (CT5efs) was used to maximize both the yields of produced virus and the peak yield of FIV pre-integration complexes in target cells.
  • CT5efs contains a 29 nucleotide insertion in the env ORF, resulting in a frame shift without deletion of any FIV sequences. This construct, therefore, permitted unbiased analysis in the present study because it abrogates Env-induced cytopathicity but does not delete any potential internal plus strand initiation sites.
  • VSV-G pseudotyped, replication-defective CT5efs particles (CT5efs(VSV-G)) produced in 293T cells were titered on Crandell feline kidney cells (CrFK) cells using immunoperoxidase staining for Gag/Pol.
  • the end-point dilution titer of unconcentrated CT5efs(VSV-G) on CrFK cells was found to be 0.9 xlO 7 / ml.
  • uninfected CrFK cells were plated at a 3:1 ratio with clironically infected CrFK cells.
  • FIV pre-integration complexes were detected by Southern blotting as a provirus- sized band in Hirt extracts of these cells. These data demonstrate that the described single-round infection assay can be used to recover FIV pre-integration complexes in diagnostic amounts from infected target cells.
  • Example 2 Detection of a plus strand discontinuity Low molecular weight (LMW) DNA was isolated by Hirt extraction from the cultures described in Example 1 at 12, 24, 42, 48 and 60 hours after infection.
  • the resulting isolated LMW DNA contained reverse-transcribed genomes lacking any integrated viral DNA.
  • Simultaneous control Hirt extracts were made from uninfected CrFK cells.
  • the extracted LMW DNA samples were digested first with Spe I (40 U/mg DNA) for one hour at 37°C in buffer containing 250 mM M NaCl, 50 mM C 2 H 3 O 2 Na, ImM ZnSO , 50mg/ml BSA.
  • the LMW DNA samples were further treated with SI nuclease (4U/mg of DNA) for one hour at 37°C in buffer containing 250 mM M NaCl, 50 mM C 2 H 3 0 2 Na, ImM ZnSO 4 , 50mg/ml BSA.
  • the resulting digested LMW DNA samples were separated on a 1.2% agarose gel by electrophoresis and analyzed by Southern blotting.
  • the probe was a 32 P labeled Bgl II- Spe I restriction fragment spanning nucleotide 6457 to 8287 of FIV env. For a description of the probe see Talbott et al, Proc. Nat. Acad. Sci. USA 86:5743-7 (1989).
  • Example 3 Primer extention analysis mapping the FIV central polypurine tract LMW DNA was extracted from infected and uninfected cells as described in Example 2. 5' to 3' primer extension was performed using lO ⁇ g LMW DNA, 25 mM of each dNTP, 10 U Taq polymerase, lO M Tris-HCl, 15mM MgCl 2 , 50 mM KC1, and 10 pmol of a 5' end-labeled primer predicted to anneal to the plus strand about 100 bp 3' of the approximate location of the FIV central polypurine tract (cPPT).
  • the sequence of the 5' end-labeled primer is 5'-ATAATAAATCCACTGTGC-3 * (SEQ ID NO: 11).
  • the primer extention reaction conditions were as follows: denaturing at 95°C for 30 seconds, annealing at 45°C for 30 seconds, and elongating at 72° for 60 seconds. The reactions were cycled 30 times. A 5 minute denaturation step at 95°C preceded the first reaction cycle. Following primer extention, the reactions were stopped with one-half reaction volume of formamide loading dye. The primer extension reactions were separated by electrophoresis on a 6% acrylamide / 7M urea gel in parallel with S anger sequencing reactions generated from the CT5 plasmid using the same end-labeled primer (SEQ ID NO: 11).
  • a single discrete stop in primer extension was seen in LMW DNA samples from infected cells but not in LMW DNA samples from uninfected control cells.
  • This stop maps the 3' boundary of a putative gap (i.e., the 5' terminus of the internally initiated D+ strand) to a G residue in a purine-rich tract ia pol (nucleotide position 4972; underlined G in: 5'-AAAAGAAGAGGTAGGA-3'; nucleotides 5-20 of the sequence set forth in SEQ ID NO: 1).
  • This identified 16 nucleotide cPPT is located centrally within the FIV genome (nucleotide positions 4959-4974).
  • the D+ strand origin at nucleotide position 4972 is 235 nucleotides 3' of the precise center of the 9474 nucleotide pro virus and 269 nucleotides 5' of the terminus of pol.
  • the cPPT is a run of mostly purines in the distal portion of the integrase gene. A comparison of this sequence with the same region in other FIV strains is shown in Table 1. An invariant pyrimidine (a thymidine) is located - 2 to the gap nucleotide, and some FIV cPPTs contain an additional pyrimidine (a C nucleotide).
  • Example 4 RACE PCR analysis mapping the FIV central termination sequence 1 ⁇ g of LMW DNA samples extracted from infected cells and from uninfected control cells as described in Example 2 was denatured at 95°C for 4 minutes and cooled on ice. The LMW DNA samples were then poly(dA) tailed by terminal deoxynucleotide transferase. The tailing reactions contained 50 units terminal deoxynucleotide transferase, 5 ⁇ M dATP, 0.75 mM cobalt chloride, 200 mM potassium cacodylate, 0.25 mg/ml BSA, and 25 mM Tris HC1, pH 6.6, in a 20 ⁇ l reaction volume. The tailing reactions were incubated at 37°C for 30 minutes.
  • the resulting poly(dA) tailed products were extracted with phenol: chloroform soamyl alcohol and then ethanol precipitated with glycogen as carrier. 6.67 ⁇ L of each precipitated DNA product was used as template in a PCR using a sense primer FIV4576 (5*-CTGGTATCTGGCAAATGGATTGC-3*; SEQ ID NO: 12) and an antisense primer with a 17 bp oligo(dT) sequence (5'-
  • the PCR mixtures contained 1.5 mM MgCl 2 , 0.2 mM dNTPs, and Platinum Taq (Gibco BRL). Following PCR, 0.1 % of the resulting PCR product was used as template in a nested PCR using either FIV4702 (5'- CTGTCTTACAATTGTTGAGTGC-3'; SEQ ID NO: 14) or FIV4674 (5'-
  • the resulting products were directly cloned without any fractionation or purification into the pCR2.1 vector using the TA cloning method (Invitrogen).
  • the cloned inserts were then sequenced on an ABI Prism 377 automated sequencer to ensure that no spurious mutations had been introduced during PCR.
  • the vectors containing cloned inserts were introduced into DH5 ⁇ bacteria by transformation. Transformed bacteria were then plated onto agar containing ampicillin such that individual ampicillin-resistant clones could be isolated 24 hours after.
  • PCR products were composed of single prominent bands that placed the site of joining of the oligod(dA) tail approximately 80- 120 nucleotides downstream of the cPPT depending on the exact site of oligo(dT) annealing to the tail.
  • a very faint band about 200 nucleotides longer than the main band, possibly representing a minor U+ strand termination, can be seen in both panels but was not detected in any sequenced clones. No products were detected in amplifications of LMW DNA from uninfected cells.
  • the precise 3' terminus of the U+ strand was determined in all 24 clones to occur at the second T nucleotide in a CA 5 T 2 sequence downstream of the cPPT.
  • These data demonstrate that the FIV genome contains a central termination sequence with a sequence of 5'-CAAAAATT-3'.
  • Example 5 - The FIV cPPT increases the efficiency of FIV vectors A 301 bp FIV region extending from 66 nucleotides upstream of the plus strand gap to 234 nucleotides downstream of the gap was amplified by PCR. The resulting amplification product was inserted into an FIV-based lentiviral vector that contained no pol sequences and only the first 311 nucleotides of the gag ORF.

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Abstract

L'invention concerne un procédé et des matériaux concernant la fabrication et l'utilisation de vecteurs lentiviraux du virus de l'immunodéficience féline (FIV) ainsi que des cellules hôtes contenant ces vecteurs.
PCT/US2002/006818 2001-03-07 2002-03-06 Lambeau d'adn central du virus de l'immunodeficience feline Ceased WO2002072756A2 (fr)

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AU2002254128A AU2002254128A1 (en) 2001-03-07 2002-03-06 Feline immunodeficiency virus central dna flap

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* Cited by examiner, † Cited by third party
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Publication number Priority date Publication date Assignee Title
WO2004036188A3 (fr) * 2002-10-18 2004-08-19 Cylene Pharmaceuticals Procedes servant a identifier des molecules antivirales ciblees vers des structures quadruplex

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