EP1071377A1 - Administration sans aiguille de formulations de polynucleotides - Google Patents

Administration sans aiguille de formulations de polynucleotides

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Publication number
EP1071377A1
EP1071377A1 EP99916610A EP99916610A EP1071377A1 EP 1071377 A1 EP1071377 A1 EP 1071377A1 EP 99916610 A EP99916610 A EP 99916610A EP 99916610 A EP99916610 A EP 99916610A EP 1071377 A1 EP1071377 A1 EP 1071377A1
Authority
EP
European Patent Office
Prior art keywords
dna
vaccine
syringe
polynucleotide
biojectortm
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP99916610A
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German (de)
English (en)
Inventor
John Donnelly
Margaret Liu
David Volkin
Adam Simon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
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Filing date
Publication date
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP1071377A1 publication Critical patent/EP1071377A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/30—Syringes for injection by jet action, without needle, e.g. for use with replaceable ampoules or carpules
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • 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
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to the use of a needleless jet injection device for the administration of polynucleotide and polynucleotide formulations for genetic vaccination, gene delivery, and gene therapy.
  • Plasmid DNA encoding hepatitis B virus surface antigen was injected into mature muscle cells with the intent of delivering the DNA using the BIOJECTORTM needleless jet injection system; see Davis et al., 1994 Vaccine 12(16):1503-1507. Levels approaching 4-fold greater antibody production were obtained as opposed to the traditional syringe/needle combination, yet when compared with the levels obtained with regenerating muscle cells, the increase was not as substantial. Further, these increases were not observed in rats.
  • Intradermal administration via a needleless injector device was attempted with a synthetic gene encoding the G protein of bovine respiratory syncytial virus (BRSV) and the resulting data compared with that obtained via a needle administration; Schrijver et al., 1998 Vaccine 16(2/3):130-134. After three immunizations into calves, antibody titres with the needleless injections were significantly higher.
  • BRSV bovine respiratory syncytial virus
  • Intramuscular injections are often the most effective means of inducing an immune response. Thus, it is desired that results akin to that obtained intradermally be obtained intramuscularly. In order to effectively use the needleless injection device to induce such an immune response intramuscularly in humans, more consistency and a greater induction of the immune system is needed. Thus, it would be desirable to identify a system that could achieve efficient transfer of polynucleotide formulations such as DNA consistently and induce stronger immune responses than that achieved with conventional needle injection into muscle.
  • This invention relates to a method of inducing an immune response to protein antigen expressed in vivo through the use of a needleless jet injection device for the administration of DNA, DNA formulations and/or other polynucleotides for genetic vaccination, gene delivery, and gene therapy.
  • This invention further relates to an improved method of inducing an immune response to a polynucleotide vaccine in an animal, preferably a human, using a needleless injector comprising introducing the vaccine
  • polynucleotide vaccine upon entry into the animal, is not present in a bolus, and wherein the polynucleotide vaccine, upon entry into the animal, is substantially evenly distributed in an area of muscle tissue.
  • This invention further relates to a method of inducing an potent immune response to a polynucleotide vaccine in an animal using a needleless injector comprising introducing the vaccine intramuscularly wherein the polynucleotide vaccine, upon entry into the animal, is not present in a bolus, the polynucleotide vaccine having spread laterally along structural tissue planes have little resistance to fluid flow, wherein the polynucleotide vaccine, upon entry into the animal, is substantially evenly distributed in an area of muscle tissue, and wherein the introduction of the vaccine is controlled by passing the vaccine through an orifice having a predetermined diameter.
  • the nucleic acid is a DNA or cDNA.
  • Particularly preferred embodiments of this invention are to the delivery of DNA which is either naked, complexed or viral.
  • Especially preferred embodiments comprise synthetic DNA molecules encoding HIV gag and synthetic DNA molecules encoding modified forms of HIV gag.
  • This invention also relates to a method of delivering DNA to an animal for purposes of gene therapy using a needleless injector comprising introducing the vaccine intramuscularly wherein the polynucleotide vaccine, upon entry into the animal, is not present in a bolus, and wherein the polynucleotide vaccine, upon entry into the animal, is substantially evenly distributed in an area of muscle tissue.
  • the amount is sufficient to effect the desired prophylactic or therapeutic response.
  • Administration by the method of this invention results in significant enhancements in antibody titers (i.e., in some instances over 100- fold) compared to conventional needle injections.
  • the method of this invention also produces a marked reduction in variability from animal to animal, and increased rates of sero-conversion (near 100%). This constitutes a surprising and significant advance over the traditional administration (i.e., via a syringe/needle combination), which leads to only a moderate response with moderate rates of sero-conversion ( ⁇ 40%) at best.
  • the administration of polynucleotides by needless jet injection results in a substantially increased distribution of injected fluid within the muscle compared to conventional
  • DNA uptake by antigen presenting cells is believed to be an extremely potent means of inducing immune responses.
  • forcing liquid into the muscle and connective tissue might induce an inflammatory response which would attract a variety of immune cells such as macrophages, dendritic cells, eosiniphils, neutrophils, etc. which could result in increased uptake of either DNA or of expressed antigen and hence lead to increased immunogenicity.
  • the variability in measured antibody response of the cohorts immunized with the needleless injector is significantly reduced relative to that of the syringe/needle.
  • the injectate is DNA.
  • DNA Especially preferred embodiments include the delivery of DNA which is naked, complexed or viral.
  • DNA may be in plasmid form, either supercoiled or not.
  • synthetic DNA molecules which encode HIV gag and synthetic DNA molecules encoding modified forms of HIV gag, en ⁇ , pol, nef, or rev genes, influenza HA, Ml, or NP genes, and herpes simplex virus (HSV) proteins such as gD and gB or papillomavirus LI protein.
  • HSV herpes simplex virus
  • FIGURE 1 illustrates the results of the gel quantification of the component of supercoiled DNA (SC) to total DNA (SC + OC) after various treatments with needle or needleless injector; see Example 2B for details.
  • FIGURES 2A and 2B illustrates a gel (Fig. 2A) and the results of gel quantification (Fig. 2B) wherein samples at two different concentrations were passed through either a No.2 (small diameter orifice) or No.5 (large
  • BIOJECTORTM syringe tip either slowly by hand in a drop- wise fashion or expelled by the burst of CO2 gas from the BIOJECTORTM; see
  • FIGURES 3A and 3B illustrates a gel (Fig. 3A) and the results of gel quantitation (Fig. 3B) wherein samples at a high concentration passed through either a No.2 (small diameter orifice) or No.5 (large diameter orifice) BIOJECTORTM syringe tip, either slowly by hand in a drop-wise fashion or expelled by the burst of CO2 gas from the BIOJECTORTM; see Example 2D for details.
  • FIGURE 4 (A) illustrates the haemagglutination inhibition (HI) responses of groups of four African green monkeys immunized by needle or BIOJECTORTM three times with 10 ⁇ g of influenza HA DNA per immunization; see Example 4A for details.
  • HI haemagglutination inhibition
  • FIGURE 4 (B) illustrates ELISA antibody responses for groups of four African green monkeys immunized by needle or BIOJECTORTM three times with 10 ⁇ g of HA DNA per immunization; see Example 4A for details.
  • FIGURE 5 (A) illustrates enhanced anti HIV-1 gag antibody titers (as measured by ELISA) in guinea pigs immunized with the BIOJECTORTM as compared to that obtained with a needle four weeks post a single dose of either 80, 400 or 2000 meg of HIV-1 gag DNA; see Example 4B for details.
  • FIGURE 5 (B) illustrates the Anti-gag antibody responses four weeks after a second immunization with the identical vaccines in the fourth week; see Example 4B for details.
  • FIGURE 6 illustrates the results after cohorts of five guinea pigs were immunized at either upper or lower hamstring sites using either a needle or BIOJECTORTM needleless syringe, with or without anesthesia; see Example 4C for details.
  • FIGURE 7 illustrates the results from the reporter gene activity of human heat resistant Secreted Alkaline Phosphatase (SeAP) measured in guinea pigs injected once with either a high or low dose of SeAP encoding plasmid DNA, either by insulin syringe or BIOJECTORTM.
  • the cohort averages are plotted as a function of time; see Example 5A for details.
  • FIGURES 8A, B and C illustrate the results from the reporter gene activity of human heat resistant secreted Alkaline phosphatase (SeAP)
  • FIGURE 9 illustrates the results from viscosity measurements of DNA in either saline or saline/glycerol at various temperatures and concentrations of DNA; see Example 2E for details.
  • “Complexed” means interacting to other matter via physical forces at close range, including but not limited to, electrostatic, van der Waals, hydrophobic and double layer, to associate or continue to interact for a time sufficient to achieve the desired function. Desired functions include but are not limited to protection from nucleases, transfection of the cell wall, endocytosis through receptor mediated pathways as well as others.
  • Polynucleotide means a nucleic acid which contains essential regulatory elements such that upon introduction into a living, vertebrate cell, it is able to direct the cellular machinery to produce translation products encoded by the genes comprising the polynucleotide.
  • PNV polynucleotide vaccine
  • Promoter refers to a recognition site on a DNA strand to which the RNA polymerase binds.
  • the promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity.
  • the complex can be modified by activating sequences termed “enhancers” or inhibiting sequences termed “silencers.”
  • Leader refers to a DNA sequence at the 5' end of a structural gene which is transcribed along with the gene.
  • the leader usually results in the protein having an N-terminal peptide extension sometimes called a pro- sequence.
  • this signal sequence which is generally hydrophobic, directs the protein into endoplasmic reticulum from which it is discharged to the appropriate destination.
  • Intron refers to a section of DNA occurring within a gene which does not code for an amino acid in the gene product. The precursor RNA of the intron is excised and is therefore not transcribed into mRNA nor translated into protein.
  • Restriction site refers to a sequence specific cleavage site of restriction endonucleases.
  • Vector refers to some means by which DNA fragments can be introduced into a host organism or host tissue. There are various types of vectors including plasmid, bacteriophages and cosmids.
  • Effective amount means sufficient PNV is injected to produce adequate levels of the transgene product. One skilled in the art recognizes that this level will vary depending on therapeutic or immuno- function desired.
  • Effective dose is an amount sufficient to produce adequate immune response in the subject to afford a protective immune response.
  • Geometric Mean Titer or “GMT” means the nth root of the product of n variables.
  • Blood refers to a roughly spherical accumulation of injectate, containing substantially the entire dose of the vaccine.
  • the needleless delivery system results in improved immunogenicity of greater than two orders of magnitude larger than naked DNA in saline or as large as over 100-fold enhancement over similar vaccination via a syringe/needle combination have been obtained after only two injections. There is also observed a near 100% seroconversion and reduced variability amongst the samples.
  • the highest antibody titers measured in an animal from a needleless injection cohort is at most 64-fold higher than the lowest titer measured, whereas the highest titer measured in a needle/syringe cohort is as much as 1,024-fold higher than the lowest titer.
  • An aspect deemed important in the present invention is use of the proper syringe tip in conjenction with the needleless injectin device. If the orifice of the syringe tip is too small or presents too large of a viscous resistance or impeadance, then the vaccine will not be injected intramuscularly. Rather, it will only penetrate the dermis, and will be present in substantially a subcutaneous bolus. Conversely, if the syringe tip is too large, or presents too small a viscous resistance or impeadance to flow, then the jet of the vaccine will penetrate too deeply, cutting a jet track in the muscle as it traverses, often striking bone or other deep tissues.
  • Another aspect of the present invention is the discovery that, at higher concentration (e.g., at 5 milligrams/ml), the nucleic acid is better protected from shear forces and is less susceptible to degradation. It has been found that there is a higher percentage of available DNA at 5 mg/ml than that found at 1 mg/ml). Therefore, one method of increasing the potency of the vaccine is to increase the concentration of polynucleotide present. This is unexpected over the prior art and further contributes to the novelty of the present invention. Thus, the present invention further entails introducing the vaccine, or DNA in the case of DNA delivery, at a concentration of at least 5 mg/ml.
  • Preferred embodiments of this invention are to the delivery of polynucleotides which are either naked, complexed or viral, with the polynucleotides being DNA or RNA.
  • any polynucleotide formulation injectable by syringe can be alternatively injected using a needleless injection device, leading to increased and enhanced biodistribution.
  • the administration of the polynucleotide formulation is via Bioject's BIOINJECTOR TM 2000 injection device, although other systems can be used.
  • the DNA or other polynucleotide chosen can be inserted into an appropriate expression vector.
  • the vector may be any known vector, including plasmids, cosmids and viral vectors which can function in the recipient cell.
  • the vectors used in any of the host cells will contain a 5' flanking sequence (also referred to as a "promoter"), whether it be a native (i.e., from the same species and or strain as the host cell), heterologous (i.e., from a different species and/or strain), or a hybrid thereof.
  • the promoter is that of cytomegalovirus (CMV).
  • the vectors may also contain other expression-control elements, such as enhancers and sequences which assist the host in expressing the peptide, such as an origin of replication element, a transcriptional termination element, a complete intron sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and/or a selectable marker element.
  • a preferred reporter gene incorporated into the vectors of the present invention is that of the human placental heat resistant secreted
  • Especially preferred embodiments comprise the administration of synthetic DNA molecules encoding HIV gag and synthetic DNA molecules encoding modified forms of HIV gag.
  • Another preferred embodiment of the present invention is the administration of DNA encoding influenza HA/georgia.
  • Other preferred embodiments include the administration of DNA encoding proteins of the herpes simplex virus (HSV), such as gB and del gD, and the human papillomavirus (HPV).
  • HSV herpes simplex virus
  • HPV human papillomavirus
  • Synthetic DNA molecules encoding HIV gag and synthetic DNA molecules encoding modified forms of HIV gag are described and claimed in co-pending patent application Serial Nos. 60/037846, 60/037854 and 09/017981, which are hereby incorporated by reference.
  • the codons of the synthetic molecules are designed so as to use the codons preferred by the projected host cell.
  • the synthetic molecules may be used as a polynucleotide vaccine which provides effective immunoprophylaxis against HIV infection through neutralizing antibody and cell-mediated immunity.
  • polynucleotide formulations suitable for use in this invention are viral particles.
  • examples include adenovirus particle formulations, adeno-associated virus (AAV) particles, and alpha virus particles.
  • AAV adeno-associated virus
  • alpha virus particles One skilled in the art will recognize others which can also be advantageously introduced by needleless injection.
  • the amount of expressible DNA or polynucleotide to be introduced into a vaccine recipient will depend on the strength of the transcriptional and translational promoters used and on the immunogenicity of the expressed gene product. In general, an immunologically or prophylactically effective dose of about 1 ng to 100 mg, and preferably about 0.1 mg to 10 mg is administered directly into muscle tissue. It is also contemplated that booster vaccinations are to be provided at, for example, monthly or later time points. Following vaccination with HIV polynucleotide immunogen, boosting with HIV protein immunogens such as gpl ⁇ O, gpl20, and gag gene products is also contemplated. Parenteral administration, such as intravenous, intramuscular, subcutaneous or other means of administration of interleukin-
  • the polynucleotide or any DNA utilized in the methods of the present invention may be naked, that is, unassociated with any proteins, adjuvants or other agents which impact on the recipients' immune system.
  • the polynucleotide or DNA is complexed or viral form.
  • the polynucleotide or other DNA is in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline.
  • the polynucleotide or DNA may be associated with liposomes, such as lecithin liposomes or cationic, anionic, or neutral lipids mixtures or other liposomes known in the art, such as a DNA-liposome mixture, or complex.
  • the DNA may be associated with an adjuvant known in the art to boost immune responses, such as a protein, aluminum salts and/or other carrier.
  • an adjuvant known in the art to boost immune responses, such as a protein, aluminum salts and/or other carrier.
  • Agents which assist in the cellular uptake of DNA such as, but not limited to, calcium ions, may also be used to advantage. These agents are generally referred to herein as transfection facilitating reagents and pharmaceutically acceptable carriers.
  • the polynucleotide used in the instant invention is administered intramuscularly, subcutaneously or both.
  • Another embodiment is a method of inducing prolonged gene expression through the injection of polynucleotide via the needleless injection system for gene therapy.
  • BIOJECTOR TM2000 needleless injection device a set of disposable syringe tips (Nos. 2, 3, 4, 5, and 7) and CO2 cartridges were purchased from Bioject, Inc. (Portland, Oregon).
  • Plasmid DNA was fermented and purified as described previously in the art. Either one of two transgenes were incorporated into the plasmids — influenza HA/georgia or HIV-1 gag driven by the CMV promoter — for use in the immunogenicity studies. VUns-HA (PR8) encoding the haemagglutinin (HA) from A/PR/8/34 was used in the influenza study. A plasmid containing the human placental heat resistant Secreted Alkaline Phosphatase (SeAP) reporter gene was constructed, also driven by the CMV promoter in the same vector backbone as the HA or gag constructs.
  • SeAP human placental heat resistant Secreted Alkaline Phosphatase
  • Each injection site received 0.5 mL of inoculum for a total dose of 10 ⁇ g of plasmid per animal.
  • Animals receiving needle injections were injected in the frontal aspect of both thighs in the largest part of the quadriceps muscle near its midpoint.
  • Animals receiving BIOJECTORTM injections were injected using the No. 3 cartridge in the anterolateral aspect of each thigh. Injections were given at the beginning of the experiment, with booster immunizations being given 6 and 18 weeks later. Blood was collected at the beginning of the experiment and at 2-4 week intervals thereafter and was assayed for antibodies to the A PR/8/34 virus by haemagglutination inhibition (HI) using chicken red blood cells with 4 HA
  • HI haemagglutination inhibition
  • Anti-gag Ab ELISA Antibody ELISA assays were performed with Nunc immunosorb 96 well plates. HIV p24 antigen protein was purchased from Intracell. Secondary antibody conjugated with the Alkaline phosphatase was purchased from Jackson Immuno Research (West Grove, PA) for the guineas pigs. BSA, Tween-20 and OPD substrate was purchased from Sigma Chemical (St. Louis, MO). iii.
  • PHOSPHA-LITETM SeAP expression level kits were purchased from Tropix (#BP300, Bedford,MA)), and reagents were prepared according to the instructions in the kit.
  • White 96-well luminometer plates were purchased from Fisher Scientific (#142-45-181). E. Instruments
  • Optical densities of the ELISA dilutions were read on a Bio Tek Instruments UV900HDi 96 well plate reader. Luminescence from the serum SeAP levels was read on a Dynex luminometer. Gel negatives were scanned on a Bio Rad GS-700 Imaging Densitometer and stored on a PC.
  • HA georgia DNA at 100 ⁇ g/ml was prepared in a stock solution.
  • the DNA was passed through various syringe tips or syringe/needle combination, into a conical tube, agarose gel, or pig skin covered agarose gel (a model for human skin on muscle). Samples were loaded into either No. 3 or 7 syringe tips, injected, and collected. DNA was then run on a gel in triplicate and the quantity of supercoiled (S.C.) and open-circle (O.C.) DNA was determined relative to a standard curve. The relative values of super-coiled to total DNA (SC + OC) was determined for each sample.
  • S.C. supercoiled
  • O.C. open-circle
  • Fig. 1 shows the results of the gel quantification of the component of supercoiled DNA (SC) to total DNA (SC + OC).
  • the control sample (DNA solution befor other treatments) is indicated on the left with a relative value of 1.000.
  • the 23g needle was statistically indistinguishable from the untreated control sample in the percentage of DNA in the supercoiled conformation.
  • incubating the DNA in a syringe tip did not degrade the DNA.
  • BIOJECTORTM syringe tip No. 2 or No. 5 Stock gag DNA at both 1 mg/ml and 5 mg/ml in normal saline was loaded into either a BIOJECTORTM syringe tip No. 2 or No. 5. It was either then expelled either slowly in a dropwise fashion by pushing on the plunger slowly by hand into an eppendorf tube, or fired from the BIOJECTORTM 2000 needleless jet injection device into a conical 50 ml disposable centrifuge tube, where it was collected and transferred to an eppendorf with a P1000 Gilson Pipetteman.
  • Samples were prepared as above and loaded onto a 1% agarose gel, run for 90 minutes at 70 V/cm, stained with 20 ⁇ g /ml Ethidium Bromide and destained for 30 min in deionized water on a rocking stage.
  • the gel negative optical density was scanned into the computer on a Bio-Rad GS-700 Imaging Densitometer with the resolution set at 42 ⁇ m and the quantitation was done using Molecular Analyst Software, Version 1.3 (Bio Rad Lab) to analyze the scanned data.
  • a number of samples were passed through either a No. 2 (small diameter orifice) or No. 5 (large diameter orifice) BIOJECTORTM syringe tip, either slowly by hand in a drop wise fashion or expelled by the burst of CO2 gas from the BIOJECTORTM.
  • Two sets of samples were run, 1 mg/ml (those on the left of the figure) and 5 mg/ml (on the right). Lane position is enumerated from left to right sequentially across the gel. In the left-most lane of each set (lanes 1 and 6), a control was run in which no
  • BIOJECTORTM syringe The effects of the BIOJECTORTM syringe on the supercoiled content of the DNA was determined by agarose gel electrophoresis of each of the ejected DNA samples. As a control, the starting plasmid DNA was also applied to the same gel. An additional control included a sample of HlV-gag
  • DNA applied to each lane of the gel for each form of DNA (SC, OC and linear).
  • the starting material (lane 1) was mostly SC DNA, with only a small quantity of OC DNA observed. Quantitation of the bands in lane 1 indicated that the starting plasmid DNA was 91% SC, 9% OC, 0% linear.
  • the results shown in lanes 2, 3 and 5 indicate that ejection of HIV-gag DNA through a 28 gauge needle or slowly through either a No. 2 or a No. 5 BIOJECTORTM syringe did not have a significant effect on the percentage of SC DNA and caused no detectable loss of material. However, in lanes 4 and 6 small amounts of linear DNA were observed in the gel, presumably due to shearing of the DNA caused by fast ejection through the BIOJECTORTM syringe.
  • Pilot experiments were conducted using Trypan Blue Stain 0.4% (Gibco # 15250-061) in order to visualize the extent of bio-distribution within the target tissue.
  • the diluent, phosphate buffer saline, pH 7.2 (PBS) was ordered from Merck Research Laboratory's internal laboratory supply group. Photographs were taken with a Nikon N70 35mm camera using Kodak elite II 400 slide film.
  • PBS phosphate buffer saline, pH 7.2
  • DNA/trypan blue was mixed either 1:1 with PBS, 200 ⁇ g/ml or 2 mg/ml gag DNA in saline, or in a 77 % ratio of DNA to trypan blue in order to achieve a final concentration of 5 mg/ml gag DNA with trypan blue.
  • DNA/trypan blue was loaded into the weakest penetrating syringe tip, a No. 2, to 200 ⁇ l following the manufacturer's instructions, tapping hard to get rid of air bubbles that may form at the interface of the plunger surface and the o-ring seal. A fresh or well charged CO2 was previously loaded into the
  • BIOJECTORTM device the syringe tip was locked into place.
  • the guinea pigs were anesthesized with a mixture of ketamine (44 mg/kg) and xylazine (5 mg/kg) and shaved of their hair in the area of the upper hamstring. Then an injection of 200 ⁇ l injectate was given to each guinea pig in the fleshy part of the upper hamstring, using a flat plastic ruler underneath the muscle tissue to support it when making the injection. The exact site of injection was determined from the anatomy of each animal. The bone that runs from the knee towards the spine was palpated. At a point two thirds the way from the knee to the spine and 4-6 mm posterior, a dot was made with a magic marker in order to help line up the BIOJECTORTM syringe tip. As per the manufacturer's instructions, firm pressure was absolutely necessary to insure the skin retention ring of the BIOJECTORTM
  • the guinea pigs were sacrificed and photographs of the closed skin, open skin, cross section of the muscle, and pool of injectate were taken.
  • BIOJECTORTM needleless jet injection device we restrained the guinea pigs by hand on a wood block and proceeded to immunize them without anesthesia An alternate site was identified, lower on the hamstring near the back of the knee joint; however, there is considerably less muscle tissue just below the skin surface and so it would be preferable for BIOJECTORTM injections to use the upper hamstring.
  • BIOJECTORTM syringe tips were loaded with DNA/trypan blue combinations at various concentrations and injected into various muscle groups of a rhesus monkey that had previously been scheduled for euthanasia for other compelling ethical considerations.
  • the rhesus was anesthesized with a dose of ketamine (10 mg/kg) .
  • ketamine 10 mg/kg
  • Bio-distribution syringe tip selection experiments were conducted on awake rabbits in their sacrospinalis back muscle.
  • the site of injection for the anterior sites are given approximately 2-3 cm below the level of the posterior rib cage.
  • the posterior sites are given approximately 2-3 cm above the iliac crest.
  • the rabbits were shaved earlier and injections were made with syringe tips ranging from No. 2 to No. 7 at DNA concentrations from 0 (PBS) to 5 mg/ml HIV gag DNA.
  • PBS DNA concentrations from 0
  • each side of the sacrospinalis muscle was dissected out and then thinly sliced to visualize the extent of the DNA/ trypan blue dye distribution.
  • Ketamine was used (10 mg/kg) to put the animals asleep.
  • a stock solution of 2 mg/mL plasmid VUns-HA (PR8) encoding the haemagglutinin (HA) from A/PR/8/34 in 0.15 N saline was diluted with sterile 0.01 M phosphate-buffered saline (pH 7.4) to a final concentration of 10 ⁇ g/mL.
  • DNA solutions were kept on ice until administered.
  • Groups of four African green monkeys of either sex weighing between 3.8 and 7.1 kg were inoculated with plasmid intramuscularly in both quadriceps muscles. Each injection site received 0.5 mL of inoculum for a total dose of 10 ⁇ g of plasmid per animal.
  • Animals receiving needle injections were injected in the frontal aspect of both thighs in the largest part of the quadriceps muscle near its midpoint. Animals receiving BIOJECTORTM injections were injected using the No. 3 syringe tip in the anterolateral aspect of each thigh. Injections were given at the beginning of the experiment, with booster immunizations being given 6 and 18 weeks later.
  • haemagglutination inhibition HI
  • ELISA whole formalin-inactivated A/PR/8/34 virus coated onto 96-well plates and developed with peroxidase- conjugated antibody to human IgG.
  • Anti-gag Ab ELISAs were measured from sera and titers assigned from the OD plate readings by endpoint titer (criteria of greater than two fold above background for a sero conversion). Typically first dilutions were 100:1 but were sometimes as high as 400:1, with serial dilutions down the plate of typically 3X or 4X. Endpoint titers were assigned where optical densities of the chromogenic agent (OPD) were greater than 2.5 times above the background level of a well. Typically, backgrounds levels on the plate were around 0.040 OD, so end-point titer thresholds were typically selected at 0.100 OD.
  • OPD optical densities of the chromogenic agent
  • guinea pigs immunized with the BIOJECTORTM revealed enhanced anti HIV-l gag antibody titers, as measured by ELISA, 4 weeks post a single dose of either 80, 400 or 2000 meg of HIV-l gag DNA.
  • the lowest dose only one of six animals sero-converted after injection with a needle, whereas four of 5 animals assayed, sero- converted after BIOJECTORTM immunization.
  • the intermediate dose only 2/6 animals seroconverted via needle injection whereas 6/6 seroconverted via needleless jet injection.
  • the cohort geometric mean titer (GMT) exhibited a ten fold enhancement at the 400 meg dose.
  • the cohort GMTs differed by a significant 80 fold enhancement.
  • Fig. 5(B) shows the Anti-gag antibody responses from the serology four weeks later after a second immunization with the identical vaccines. All BIOJECTORTM immunized cohorts in this experiment have 100% seroconversion, whereas the needle injected cohorts do not. At the low dose, the BIOJECTORTM conferred a 12 fold enhancement in Ab titer, while at the intermediate dose it conferred a 144 fold enhancement. This unusually high
  • - 23 - enhancement is slightly exaggerated in that it appears as if three guinea pigs did not respond at all after needle immunization at the intermediate dose, thus depressing the needle GMT and thus boosting the relative enhancement factor.
  • Anti-gag Ab ELISAs were measured from sera and titers assigned from the OD plate readings by endpoint titer (criteria of more than two foldabove background for a sero conversion). Typically first dilutions were 100:1 but were sometimes as high as 400:1, with serial dilutions down the plate of typically 3X or 4X. Endpoint titers were assigned where optical densities of the chromogenic agent (OPD) were greater than 2.5 times above the background level a well. Typically, backgrounds levels on the plate were around 0.040 OD so end-point titer thresholds were typically selected at 0.100 OD.
  • OPD optical densities of the chromogenic agent
  • Cohort 1 received a total dose of 5 mg of DNA via 25g5/8 needle while under anesthesia.
  • Cohort 2 received a total dose of 5 mg of DNA via BIOJECTORTM with No. 3 syringe tip while under anesthesia.
  • Cohort 3 received a total dose of 5 mg of an HIV-l gag DNA construct that lacked a leader sequence (DNA #80) via BIOJECTORTM with No. 3 syringe tip while under anesthesia.
  • Cohort 4 received a total dose of 1 mg of DNA via 25g5/8 needle while under anesthesia.
  • Cohort 5 received a total dose of 1 mg of DNA via BIOJECTORTM with No. 3 syringe tip while under anesthesia.
  • Cohort 6 received a total dose of 1 mg of DNA via BIOJECTORTM with No. 3 syringe tip while awake, temporarily restrained by a collar during chair training.
  • SeAP experssion levels detected in the blood were determined following the manufactuer's instructions. Standard curves of Alkaline phosphatase in pooled guinea pig serum were used to quantitativly determine the quantity of active SeAP protein circulating in the guinea pigs.
  • results are presented of the reporter gene activity of SeAP which was measured in guinea pigs injected once with either a high or low dose of SeAP encoding plasmid DNA, either by insulin syringe or BIOJECTORTM.
  • the cohort averages are plotted as a function of time.
  • the SeAP levels in both needle cohorts started low and increased over the first week as indicated by the solid circle and square.
  • the cohort injected via BIOJECTORTM did not demonstrate a large response (solid diamond) whereas the high dose BIOJECTORTM cohort exhibited a 3 fold enhancement in SeAP activity at day 2, but steadily falls off during the first week.

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Abstract

L'invention a trait à un procédé permettant de provoquer une réaction immunitaire contre un antigène protidique exprimé in vivo, au moyen d'un dispositif d'injection à pression sans aiguille servant à administrer de l'ADN, des formulations d'ADN et/ou d'autres polynucléotides en vue de mettre en oeuvre une vaccination génétique, une administration de gène ou une thérapie génique.
EP99916610A 1998-04-14 1999-04-12 Administration sans aiguille de formulations de polynucleotides Withdrawn EP1071377A1 (fr)

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CA2358385C (fr) 1998-12-31 2013-08-06 Chiron Corporation Polynucleotides codant pour des polypeptides antigeniques du type c du vih; polypeptides et leurs utilisations
EP2412242A3 (fr) 2001-07-05 2012-06-13 Novartis Vaccines and Diagnostics, Inc. Polynucléotides codant pour des polypeptides antigènes de type C du VIH, polypeptides et leurs utilisations
EP2172552A3 (fr) 2001-10-11 2010-07-21 Merck Sharp & Dohme Corp. Acide nucléique recombinant comprenant des régions de AD6
JP4475561B2 (ja) 2001-10-11 2010-06-09 メルク・シャープ・エンド・ドーム・コーポレイション Ｃ型肝炎ウイルスワクチン
JP4512761B2 (ja) * 2003-12-03 2010-07-28 雪生 ▲高▼橋 動物管理システム
EP2570423B1 (fr) 2005-06-17 2023-05-03 MSD Italia S.r.l. Vaccin à acide nucléique contre l'hépatite C
JP2017000667A (ja) 2015-06-16 2017-01-05 国立大学法人三重大学 無針注射器及びそれを用いた注射対象領域へのｄｎａ導入方法
JP7280203B2 (ja) * 2018-02-09 2023-05-23 株式会社ダイセル 注入器及びそれを用いた注入対象の細胞核内への生体分子を含む溶液の注入方法
CN111699011B (zh) * 2018-02-09 2023-07-28 株式会社大赛璐 注入器
JP7280202B2 (ja) * 2018-02-09 2023-05-23 株式会社ダイセル 注入器及びそれを用いた注入対象への生体分子を含む溶液の注入方法
CN114748614A (zh) * 2021-12-27 2022-07-15 上海药明生物医药有限公司 一种利用无针注射器进行动物免疫的方法
WO2023204264A1 (fr) * 2022-04-20 2023-10-26 株式会社ダイセル Composition pharmaceutique liquide

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US4790824A (en) 1987-06-19 1988-12-13 Bioject, Inc. Non-invasive hypodermic injection device
US4940460A (en) 1987-06-19 1990-07-10 Bioject, Inc. Patient-fillable and non-invasive hypodermic injection device assembly
US5064413A (en) 1989-11-09 1991-11-12 Bioject, Inc. Needleless hypodermic injection device
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IL113817A (en) * 1994-06-30 2001-03-19 Merck & Co Inc Polynucleotide for vaccination against the umbilical cord virus
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