JP2002511396A - Needleless administration of polynucleotide preparations - Google Patents

Abstract

  (57) [Summary] The present invention relates to protein antigens expressed in vivo by the use of needle-free injection devices for the administration of DNA, DNA preparations and / or other polynucleotides for genetic vaccination, gene delivery and gene therapy. A method of inducing an immune response.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of needleless injection devices for the administration of polynucleotides and polynucleotide preparations for genetic vaccination, gene delivery and gene therapy.

BACKGROUND OF THE INVENTION Traditionally, injection of pharmaceutical or vaccine formulations into humans has been by syringe / needle combination. While this is an effective means of transportation, the risks associated with needles (
For example, pathogens carried by blood) are prominent. In addition, needles place an additional burden on both children, especially for small children, as awkward needle stings. With the advent of advanced needle-free injection devices such as Bioject's BIOJECTOR ™ 2000, intramuscular injection of substances with higher efficacy, less persistent skin damage and reduced risk of transmission of blood-borne pathogens (US Pat. Nos. 5,383,851, 5,064,413)
Nos. 4,940,460, 4,790,824 and 4,596,556).

[0003] This system for gene transfer using a propellant injection technique (particularly a PED-O-JET ™ syringe) to introduce a reporter gene (eg, chloramphenicol acetyltransferase (CAT)). An experiment was performed to assess the efficacy of (see Furth et al., 1992 Analytical Biochem. 205: 365-368). However, not all samples were able to detect CAT activity, and moreover, those samples showed variability. The use of such techniques in generating an immune response has not been tested.

[0004] Subsequently, experiments in the field of DNA-based immunization were performed. Plasmid DNA encoding the hepatitis B virus surface antigen was injected into mature myocytes with the intention of delivering it by the BIOJECTOR ™ needle-free injection system (Davis et al., 1994 V).
accine 12 (16): 1503-1507). Although antibody production levels were nearly four times higher than those obtained with a conventional syringe / needle combination, the increase was not significant when compared to levels obtained with regenerating muscle cells. Furthermore, these increases were not observed in rats.

[0005] Intradermal administration with a needle-free injection device has been attempted with a synthetic gene encoding the G protein of bovine respiratory syncytial virus (BRSV) and the data obtained are compared to those obtained with needle administration. (Schrijver et al., 1998 Vaccine 16 (2/3): 130-134). After three immunizations of calves, antibody titers were significantly higher with needleless injection.

[0006] Intramuscular injection is often the most effective means of inducing an immune response. Therefore, it is desirable that the same results as obtained by intradermal administration be obtained by intramuscular administration. Effective use of needle-free injection devices to induce such immune responses in humans by intramuscular administration requires a more consistent and greater induction of the immune system. Therefore, it would be desirable to identify systems that consistently achieve efficient introduction of a polynucleotide formulation, such as DNA, and that can elicit a stronger immune response than with conventional needle injection into muscle.

SUMMARY OF THE INVENTION [0007] The present invention relates to DNA, D, for genetic vaccination, gene delivery and gene therapy.
A method for inducing an immune response to a protein antigen expressed in vivo by use of a needle-free injection device to administer a NA formulation and / or other polynucleotides.

[0008] The present invention is further directed to an improved method of inducing an immune response to a polynucleotide vaccine in an animal, preferably a human, using a needleless injector, the method comprising introducing the vaccine intramuscularly. Comprising, when the polynucleotide vaccine enters the animal, is not present in the bolus; and, when the polynucleotide vaccine enters the animal, substantially uniformly within the area of muscle tissue. A method characterized by being distributed.

[0009] The present invention is further directed to a method of inducing a potent immune response against a polynucleotide vaccine in an animal using a needle-free injector, the method comprising introducing the vaccine intramuscularly. When the vaccine enters the animal,
That the polynucleotide vaccine, which is not present in the bolus and extends laterally along the surface of the structural tissue, has little resistance to flow; when the polynucleotide vaccine enters the animal, muscle tissue And a method wherein the introduction of the vaccine is controlled by passing the vaccine through an orifice having a predetermined diameter.

[0010] In a preferred embodiment, the nucleic acid is DNA or cDNA. A particularly preferred embodiment of the present invention is the delivery of naked or complexed or viral DNA.
Particularly preferred embodiments include synthetic DNA molecules encoding HIV gag and synthetic DNA molecules encoding modified forms of HIV gag.

The present invention also provides a method of delivering DNA to an animal for gene therapy using a needle-free injector, comprising introducing the vaccine intramuscularly, wherein the polynucleotide vaccine is When entering the animal, it is not present in the bolus;
And a method wherein the polynucleotide vaccine is substantially uniformly distributed within the area of muscle tissue upon entry into the animal. Preferably, the amount is sufficient to produce the desired prophylactic or therapeutic response.

Administration according to the method of the present invention results in a significant increase in antibody titer (compared to normal needle injection).
Ie, in some cases 100 times). The method of the present invention also results in a marked reduction in inter-animal variability and increased seroconversion (about 100%). This constitutes a surprising and significant advance over conventional dosing (i.e., with a syringe / needle combination) that produces at most a modest response of seroconversion (-40%). Administration of a polynucleotide by needleless injection results in a substantially increased intramuscular distribution of the injection fluid as compared to normal needle / syringe delivery. The propellant of the polynucleotide formulation, after entering the body, appears to advance transversely to the axis of the injection, crossing the structural surface of the tissue, such as the fascia. These planes tend to have a reduced level of resistance, so that the liquid jets of the vaccine tend to travel along them. Without wishing to be bound by theory, increased intramuscular distribution of the polynucleotide is probably necessary for induction of an improved immune response. Some theories could explain how increasing the distribution improves the immune response. Increasing the distribution of the vaccine throughout the muscle could give more antigen presenting cells (eg, macrophages and dendritic cells that take up DNA). Because these cells are only found at low density in muscle tissue. Uptake of DNA by antigen presenting cells is considered to be a very effective means for inducing an immune response. Alternatively, pushing fluid into muscle or connective tissue can lead to increased uptake of DNA or expressed antigens, and thus may cause increased immunogenicity, such as macrophages, dendritic cells, eosinophils, neutrophils, etc. An inflammatory response that attracts immune cells can be induced. Furthermore, the variability of the measured antibody response of the cohort immunized with the needle-free injector is significantly lower compared to the syringe / needle case.

In a preferred embodiment, the injectable substance is DNA. Particularly preferred embodiments include delivery of naked or complexed or viral DNA. The DNA may be in supercoiled or other plasmid form. Particularly preferred is HI
V gag, env, pol, nef or rev gene, influenza HA, M1 or NP gene and a herpes simplex virus (HSV) protein, such as gD and gB, or a papillomavirus L1 protein, a synthetic DNA molecule.

With respect to the following terms used throughout the specification and claims, the following definitions apply.

“Complexation” refers to short-range physical forces (electrostatic, van der Waals, hydrophobic and bilayer) that associate or maintain interactions for a time sufficient to achieve a desired function. And interaction with other substances through Desired functions include, but are not limited to, nuclease protection, cell wall transfection, endocytosis via a receptor-mediated pathway, and the like.

A “polynucleotide” contains essential regulatory elements that, when introduced into a living vertebrate cell, can direct the cellular apparatus to produce a translation product encoded by the gene containing the polynucleotide. Means nucleic acid.

“PNV” is the name given to a polynucleotide vaccine.

“Promoter” refers to a recognition site on a DNA strand to which RNA polymerase binds. The promoter forms an initiation complex with the RNA polymerase to initiate and drive transcriptional activity. The complex may be modified by activating a sequence termed "enhancer" or by suppressing a sequence termed "silencer".

“Leader” means a DNA sequence at the 5 ′ end of a structural gene that is transcribed with the gene. The leader usually provides a protein with an N-terminal peptide extension, sometimes called a prosequence. In proteins that are defined to secrete into the extracellular medium or membrane, this signal sequence, which is generally hydrophobic, directs the protein into the endoplasmic reticulum, where it is released to the appropriate destination. You.

“Intron” refers to a DNA fragment found in a gene that does not encode an amino acid in the gene product. Since the intron precursor RNA is excised, it is not transcribed into mRNA or translated into protein.

“Restriction site” means a sequence-specific site for a restriction endonuclease.

“Vector” means any means that enables a DNA fragment to be introduced into a host organism or host tissue. Various types of vectors exist, including plasmids, bacteriophages and cosmids.

“Effective (effective) amount” means injecting sufficient PNV to produce the appropriate level of transgene product. It is understood by those skilled in the art that this level can vary depending on the desired therapeutic or immune function.

An “effective (effective) dose” is an amount sufficient to provide a suitable immune response in a subject that provides a protective immune response.

“Geometric mean titer” or “GMT” means the nth root of the product of n variables.

“Bolus” means an approximately spherical injectate deposit that contains substantially the entire dose of the vaccine.

One embodiment of the present invention is that the needle-free delivery system results in improved immunogenicity by two orders of magnitude over naked DNA in saline, or by a syringe / needle combination after only two injections. A 100-fold increase over similar vaccinations. Seroconversion close to 100% and reduced sample-to-sample variability are also noted. Figure 5
In (B), the highest antibody titer measured in animals from the needle-free injection cohort is at most 64 times higher than the lowest titer measured, but the highest measured in the needle / syringe cohort. The titer is 1,024 times higher than the lowest titer.

An embodiment considered important in the present invention is the use of a suitable syringe tip with a needle-free injection device. If the syringe tip orifice is too small,
Or if it shows too much viscous resistance or impedance, the vaccine will not be injected intramuscularly. Rather, it will penetrate the dermis and will be substantially within the subcutaneous bolus. Conversely, if the syringe tip is too large,
Or, if it exhibits viscous drag or impedance that is too low for flow, the vaccine propellant penetrates too deeply and cuts the propulsion trajectory in the muscle as it traverses, often in bone or other deep areas. Reach the organization.

Another embodiment of the present invention provides that at higher concentrations (eg, 5 mg / ml)
It is the finding that nucleic acids are better protected from shear and less susceptible to degradation. At 5 mg / ml a higher percentage of available DNA than found at 1 mg / ml
Was found to exist. Thus, one way to increase the efficacy of a vaccine is to increase the concentration of polynucleotide present. This is unexpected from the prior art and contributes to the novelty of the present invention. Thus, the present invention further comprises introducing the vaccine (or, in the case of DNA delivery, DNA) at a concentration of at least 5 mg / ml.

A preferred embodiment of the present invention is the delivery of a naked or complex or viral polynucleotide, wherein the polynucleotide is DNA or RNA. Essentially, any polynucleotide formulation that is injectable by syringe, instead,
Injections can be made using a needle-free injection device that provides increased and improved biodistribution. Preferably, administration of the polynucleotide formulation is via a Bioject BIOINJECTOR® 2000 injection device, although other systems can be used.

[0031] The selected DNA or other polynucleotide can be inserted into a suitable expression vector. The vector may be any known vector, including plasmid, cosmid, and viral vectors that can function in recipient cells. Typically, the vector used in any of the host cells is native (ie,
5 ′ flanking sequences (also referred to as “promoter”), whether derived from the same species and / or strain as the host cell, or heterologous (ie, from a different species and / or strain), or hybrids thereof. Will be included).
Preferably, the promoter is of cytomegalovirus (CMV). If desired, the vector may also contain other expression control elements, such as enhancers, and sequences that assist the host in expressing the peptide, such as origin of replication elements, transcription termination elements, complete intron sequences containing splice donor and acceptor sites. , A signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding the expressed polypeptide, and / or a selectable marker element. A preferred reporter gene contained within the vector of the present invention is human placental thermostable secreted alkaline phosphatase (SeAP)
Of the gene. If the element is not present in the vector used,
They can be obtained individually and ligated into the vector. The methods used to obtain these factors are well known to those skilled in the art.

A particularly preferred embodiment involves the administration of a synthetic DNA molecule encoding HIV gag and a synthetic DNA molecule encoding a modified form of HIV gag. Another preferred embodiment of the present invention is the administration of DNA encoding influenza HA / georgia. Other preferred embodiments include DNA encoding proteins of the herpes simplex virus (HSV) (eg, gB and del gD) and human papillomavirus (HPV).
Including the administration of

Synthetic DNA molecule encoding HIV gag and synthetic D encoding modified form of HIV gag
NA molecules are disclosed in co-pending patent application Ser. No. 60/037846,
Nos. 60/037854 and 09/017981 are claimed and claimed. Preferably, the codons of the synthetic molecule are designed to use the desired codon for the intended host cell. The synthetic molecules can be used as polynucleotide vaccines to provide effective immunoprotection against HIV infection via neutralizing antibodies and cellular immunity.

[0034] Other polynucleotide formulations suitable for use in the present invention are viral particles. Specific examples include adenovirus particle preparations, adeno-associated virus (AAV) particles and alphavirus particles. One skilled in the art will recognize others that may be advantageously introduced by needleless injection.

The amount of expressible DNA or polynucleotide introduced into the vaccine recipient will depend on the strength of the transcription and translation promoters used and the immunogenicity of the expressed gene product. Generally, an immunologically or prophylactically effective dose of about
1 ng to 100 mg, preferably about 0.1 mg to 10 mg, is administered directly into the muscle tissue. It is also contemplated, for example, to provide booster vaccinations at monthly or later time points. HIV
It is also contemplated that after vaccination with a polynucleotide immunogen, boosting with an HIV protein immunogen, such as the gp160, gp120 and gag gene products. PNV of the present invention
It may also be advantageous to carry out parenteral administration (eg, intravenous, intramuscular, subcutaneous or other means of administration) of the interleukin 12 protein simultaneously with or after the parenteral introduction of.

The polynucleotide or any DNA used in the method of the present invention may be naked (
That is, without any protein, adjuvant or other substance that affects the immune system of the recipient). Alternatively, the polynucleotide or DNA is complexed or in a viral form. Preferably, the polynucleotide or other DNA is in a physiologically acceptable solution, such as, but not limited to, sterile saline or buffered saline. Or,
The polynucleotide or DNA may be associated with a liposome, such as a lecithin liposome, or a cationic, anionic or neutral lipid mixture or other liposome known in the art, such as a DNA-liposome mixture or complex. Good. Further, the DNA may be associated with adjuvants known in the art to enhance the immune response, such as proteins, aluminum salts and / or other carriers. Also, substances that assist cells in taking up DNA (eg, but not limited to calcium ions) can be advantageously used. In the present invention, these substances are collectively referred to as a transfection promoting reagent and a pharmaceutically acceptable carrier.

Preferably, the polynucleotide used in the present invention is administered intramuscularly, subcutaneously or both.

[0038] Another embodiment is a method of inducing sustained gene expression by injection of a polynucleotide with the needle-free injection system for gene therapy.

The following non-limiting examples are provided to further illustrate the present invention.

Example 1material  A. Needleless injection device BIOJECTOR (trademark) 2000 needleless injection device, one set of disposable syringe tips (No. 2
, 3,4,5 and 7) and CO_TwoInsert cartridges into Bioject, Inc. (Protland, Oregon
) Purchased from.

B. Animals Animals were purchased from Charles River, Covance or Jackson Laboratory and housed and handled according to IACUC guidelines for the care and use of laboratory animals. About 2
Purchase 50 grams of Duncan Hartley guinea pigs from Covance and for at least 2 weeks
The animal facility was adapted. Approximately 3 kg of New Zealand White rabbits were purchased from Covance and African green monkeys and rhesus monkeys were obtained from Charles River (Key Lois, FL) and New Iberia Research Center, New Iberia, LA. Anesthetics ketamine and rhompon were purchased from Phoenix Scientific (St. Joseph, MO).

C. DNA Fermentation of plasmid DNA (fer) as described in the art.
mented) and purified. Either of the two transgenes was incorporated into a plasmid (influenza HA / georgia or HIV-1 gag driven by the CMV promoter) for use in immunogenicity studies. Hemagglutinin from A / PR / 8/34 (
HA) encoding V1Jns-HA (PR8) was used in influenza studies. Similarly H
Plasmids containing the human placental thermostable secreted alkaline phosphatase (SeAP) reporter gene driven by the CMV promoter in the same vector backbone as the A or gag constructs were constructed.

D. Assay reagent i. HA / georgia hemagglutination inhibition: Plasmid V1Jns-HA (PR8) encoding hemagglutinin (HA) from A / PR / 8/34 (
2 mg / mL) in 0.15 N saline in sterile 0.01 M phosphate buffered saline (pH
It was diluted to a final concentration of 10 μg / mL in 7.4). DNA solutions were kept on ice until administration. Groups of four African green monkeys of both genders weighing 3.8-7.1 kg were inoculated intramuscularly with plasmid in both quadriceps. Each injection site received 0.5 mL of the inoculum so that the total dose of plasmid per animal was 10 μg. Animals receiving needle injections were injected into the maximal area near the midpoint of the quadriceps in both anterior thighs. Animals receiving injections with BIOJECTOR ™ were injected into the anterior lateral part of each thigh using a No. 3 cartridge. Injections were performed at the beginning of the experiment and boosts were given 6 and 18 weeks later. Blood was collected at the beginning of the experiment and at intervals of 2-4 weeks thereafter, with 4 HA units of A / PR /
A / PR / by hemagglutination inhibition (HI) using chicken erythrocytes with 8/34 virus and by ELISA using whole formalin-inactivated A / PR / 8/34 virus coated on 96-well plates. Assayed for antibodies to 8/34 virus and developed with peroxidase conjugated antibodies to human IgG.

Ii. Anti-gag Ab ELISA: Antibody ELISA assays were performed in Nunc immunoadsorbed 96-well plates. HIV p24 antigen protein was purchased from Intracell. Secondary antibodies for guinea pigs conjugated to alkaline phosphatase were purchased from Jackson Immuno Research (West Grove, PA). BSA, Tween-20 and OPD substrate were purchased from Sigma Chemical (St Louis, MO).

Iii. SEAP expression level: PHOSPHA-LITE (trade name) SeAP expression level kit was added to Tropix (# BP300, Bedford, M
The reagent was purchased from A) and the reagent was prepared according to the instructions of the kit. White 96-well luminometer plates were purchased from Fisher Scientific (# 142-45-181).

E. Instrument The optical density of the ELISA dilution was read on a BIO Tek Instruments UV900HDi 96 well plate reader. Luminescence from 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.

Example 2An in vivo test to determine the integrity of polynucleotides after needleless injection. Characterization in b  A. Red pigment on pig skin: Physical properties to assess the suitability of needle-free injection for polynucleotide formulations
The research was done. To visualize and model needle-free injection into human tissue
In a series of experiments, the aqueous phenol red dye solution was placed in a 20 CC syringe.
Inject, close the luer lock end, and place 5% agarose on the syringe body
The gel was filled. To visualize events during injection through human skin
Skin (purchased in a supermarket) as a model system with the needleless syringe tip
It was placed between agarose gel filled syringes.

In the photograph taken at that time, it was vertically aligned vertically above the syringe
BIOJECTOR ™ was recognized, and a red dye was clearly visualized in the syringe tip just before pressing the brake on the needle-free injection device. In another image, the trajectory and profile of the phenol red red dye upon entering the agarose cylinder was found to be very narrow. Because it emerged from the syringe tip and slowly expanded its traversal to the axis of propagation to about three times its initial parallel diameter. A piece of porcine skin was placed between a BIOJECTOR ™ chip and the agarose cylinder to simulate what happened to the DNA as it passed through the human skin. The profile widened shortly after it passed through the pig's skin and appeared to be turbulent, as if the openings in the skin pores created a vortex-generating surface. It was as if the injection penetrated more in the presence of pig skin than in its absence. While not wishing to be bound by theory, it is concluded that this divergent jet facilitates finding structural surfaces that have little resistance, in which case the polynucleotide formulation may be widely spread. Can be

B. Gel after passage at 100 mcg / ml 100 μg / ml HA georgia DNA was prepared in a stock solution. The DNA was passed through various syringe tips or syringe / needle combinations into conical tubes, agarose gels or agarose gels (models of human skin over muscle) covered with porcine skin. Samples were loaded into No. 3 or 7 syringe tips, injected and collected. The DNA was then run on the gel in triplicate and the amount of supercoiled (SC) and open circular (OC) DNA was measured relative to a standard curve. The relative value of the supercoiled body to the total DNA (SC + OC) was determined for each sample.

FIG. 1 shows the results of gel quantification of supercoiled DNA (SC) components relative to total DNA (SC + OC). A control sample (another pre-treatment DNA solution) is shown on the left as having a relative value of 1.000. The 23 g needle was not statistically distinguishable from the untreated control sample in the percentage of DNA in the supercoiled conformation.
As another control, incubation of the DNA in a syringe tip
Did not decompose. When the DNA was fired from a No. 7 syringe tip into a conical tube (with little or no frictional resistance in air), about 67% of the DNA was recovered in the original supercoal form, while using the same tip However, when injected into a 5% agarose gel, only about 45% remained, indicating that 55% had been cleaved back to an open or linear conformation. It is clearly evident that the percentage of supercoiled DNA did not change with further passage through the porcine skin layer, but the error bars were larger, indicating greater variability in triplicate measurements. Is shown. Finally, the DNA is not removed from a No. 7 syringe tip (having a larger diameter orifice) but from a No. 3 tip (having a smaller diameter orifice that gives a greater shear modulus and less permeability). It should be noted that when injected into the agarose, the percentage of supercoil dropped from 45% to nearly 32%. Thus, at this low DNA concentration (100 mcg / ml), the shear force at the outlet of the syringe tip appears to be responsible for the shear modulus for the DNA.

C. Gel-Comparison of rates at two different concentrations BIOJECTOR (stock gag DNA at both 1 mg / ml and 5 mg / ml concentrations in saline)
(Trade name) Syringe tip No. 2 or No. 5 was loaded. Then,
Slowly push the plunger slowly by hand into the Eppendorf tube slowly by dropping, or 50 ml from the BIOJECTOR® 2000 needleless injection device
Fired into a disposable conical centrifuge tube, collected in the tube and transferred to an Eppendorf equipped with a P1000 Gilson Pipetteman.

Samples were prepared as described above, loaded on a 1% agarose gel,
Moved at 0 V / cm for 90 minutes, stained with 20 μg / ml ethidium bromide, and destained in deionized water for 30 minutes on a rocking platform.

The gel was placed in a photodocumentation setup and photographed on Polaroid film which provided both positive and negative photographic emulsions. After washing the negatives in water for at least several hours, they were exposed to UV light for 10-30 seconds. Development was performed according to the manufacturer's instructions for the film.

The gel negative optical density was set to a Bio-Rad GS700 Imaging D set to a resolution of 42 μm.
Scanning into a computer on an ensitometer and quantification was performed using Molecular Analyst Software, Version 1.3 (Bio Rad Lab) to analyze the scanned data.

As shown in FIG. 2, a large number of samples were slowly dropped by hand or BIOJECTOR
The injection by ejection of CO ₂ gas from (trade name), No.2 (orifice larger diameter) (small diameter orifice) or No.5 Biojector (TM) was passed through a syringe tip. Two sets of samples were transferred, 1 mg / ml (left side of the figure) and 5 mg / ml (right side). Lane positions are sequentially numbered from left to right across the gel. In the leftmost lane of each set (lanes 1 and 6)
Untreated controls were moved. In lanes 2 and 3, DNA was loaded into a No. 2 syringe tip slowly by hand or faster by CO ₂ squirting from BIOJECTOR®. There appears to be no overall difference in the gel pattern, but there is a slight "tail" running in front of the supercoiled band. In lanes 4 and 5, the same was done with a No. 5 syringe tip. In this case, the slower sample (by hand) appears to be the same as the control, while the BI
The sample jetted with OJECTOR ™ shows a clear stain tail running in front of the supercoil band.

Looking at the same experiment performed at 5 mg / ml DNA in the set of lanes on the right, there is a “tail” in lanes 8 and 10, which are less pronounced at more viscous, higher DNA concentrations . Therefore, more viscous and / or higher DNA
The concentration seems to protect the DNA from shear. In addition, at the more viscous, higher concentrations, the greater the shear force, the greater the diameter of the syringe tip (No. 5).
Has occurred within.

D. Gel-Comparison of Rates at High Concentration Gel runs were performed with 5.5 mg / ml HIV-gag DNA in saline (each vial contains 0.8 ml of DNA solution).

The effect of a BIOJECTOR® syringe on the supercoil content of the DNA was
Judgment was made by agarose gel electrophoresis of each injected DNA sample. As a control, the starting plasmid DNA was applied to the same gel. Additional controls included a sample of HIV-gag DNA injected from a 28 gauge needle. Aliquots of each DNA sample containing 18 ng of DNA were applied to separate lanes on a 1% agarose gel. After electrophoresis, the gel was stained with bromide bromide and photographed under UV light. The negative of the gel photo was then scanned using a Bio-Rad GS-700 densitometer. The amount of DNA in each band on the gel was determined on the basis of the band intensity generated by known amounts of supercoiled (SC), open circular (OC) and linear DNA standards applied to the same gel, Molecular Measured using the Analyst (ver. 1.3) software program (Bio-Rad). To ensure accurate results, a standard curve was generated using the band intensity of the DNA standard and the mass of DNA applied to each lane of the gel for each DNA form (SC, OC and linear). A quadratic fit to the data for each of the standard curves (SC, OC and linear). The correlation coefficients of all standard curves were> 0.99.

To determine the effect of injection of plasmid DNA from a BIOJECTOR ™ syringe on the supercoil content of the DNA, 200 mcL (No. 2 syringe tip)
Or No. 2 or No. 5 filled with 500 mcL of HIV-gag DNA (No. 5 syringe tip)
5 Supercoiled plasmid DNA of 5.5 mg / ml in saline was injected from BIOJECTOR (trade name). The effect of a BIOJECTOR® syringe on the supercoil content of the DNA was determined by agarose gel electrophoresis.

Agarose gel electrophoresis and quantification of plasmid DNA injected from a Biojector syringe or 28 gauge needle is shown in FIG. The positions where SC, OC and linear HIV-gag DNA migrated on the gel are indicated on the left side of the gel. Lane 1 shows the starting DNA control. Lane 2 shows DNA after injection from a 28 gauge needle. Lanes 3 and 4 show DNA after slow hand (lane 3) or injection with a Biojector device (lane 4) from a No. 2 Biojector syringe. Lanes 5 and 6 are slow by hand (lane 5)
Or after injecting from No.5 Biojector syringe with Biojector device (lane 6)
Indicates DNA.

The starting material (lane 1) was mostly SC DNA and only a small amount of OC DNA was found. The quantification of the band in lane 1 was performed using 91% SC, 9% O
C, 0% linear. The results shown in lanes 2, 3 and 5 show that H
Slow injection of IV-gag DNA from a 28 gauge needle or from a No. 2 or No. 5 BIOJECTOR ™ syringe has no significant effect on the percentage of SC DNA and causes a detectable loss of material. It is not shown. However, in lanes 4 and 6, a small amount of linear DNA was found in the gel, probably due to BIOJECTOR (
This may be due to shearing of the DNA caused by the rapid injection from the syringe. The quantification of the DNA band in lane 4 showed that the DNA was 93.32% SC, 5.91% OC and
0.78% linear. These results indicate that a small amount of linear DNA
It is suggested that this was caused by the conversion of DNA to linear DNA. Quantitation of the DNA band in lane 6 showed that the DNA was 93.08% SC, 5.70% OC and 1.22% linear. These results also indicate a small amount of conversion of the OC DNA to linear DNA during rapid squirting from a BIOJECTOR ™ syringe. Quantitation of the DNA band in all lanes indicated complete recovery of the DNA from the needle and BIOJECTOR® syringe. In summary, injection of plasmid DNA from a No. 2 or No. 5 BIOJECTOR ™ syringe does not cause any loss of SC DNA, but only a slight conversion of OC to linear DNA. Since the starting DNA contains small amounts of OC DNA, the amount of linear DNA produced is about 1% of total DNA and should have no detectable effect on the biological efficacy of the propellant .

E. Effect of Shearing Force on Stability of Plasmid DNA Various concentrations of DNA plasmid in saline or saline / glycerol were subjected to different shearing forces using a Carri-Med Rheometer with cones and plates. The shear applied to the DNA in these samples was the largest that could be applied with a Rheometer. The best could be done to simulate a needleless injection device. Plasmid DNA was tested at 25, 250 and 2500 mcg / mL in saline. 25 and 25
Only a concentration of 0 mcg / mL was tested in saline / glycerol solution. The reason for the addition of glycerol is bulk DNA (2.5 mg / mL) to prevent shearing of low dose DNA concentrations
Was to increase the viscosity to. The samples were sheared at 10 and 25 ° C. in the presence and absence of glycerol. In the control sample, the slab (containing the sample) was lifted to the cone without rotation, and thus no shear was applied.

FIG. 9 shows the results. Conversion of supercoiled DNA to open circular DNA was determined by agarose gel electrophoresis to determine if the sample had been sheared. In summary, the data show that when sheared at 25 ° C. and diluted in saline, some shear of the DNA occurred in DNA plasmid samples at low concentrations (25 mcg / mL). Is shown. However, the same sample in glycerol / saline was not sheared. These results suggest that there may be a viscous effect on DNA shear. In addition, there is also a concentration effect, most likely because 25 mcg / mL is more sensitive to shear than samples with higher DNA concentrations. Therefore,
This data supports the notion that higher DNA concentrations, and therefore higher viscosities, protect DNA injected from needle-free injection devices.

Example 3Syringe tip suitable for skin / fascia / muscle composition of given species and injection site Biodistribution syringe tip selection study to determine the  0.4% trypan blue dye to visualize the degree of biodistribution in the target tissue
(Gibco # 15250-061) was used for pilot experiments. Phosphorus as a diluent
Acid buffered saline (pH 7.2 (PBS)) was supplied to Merck Research Laboratory's internal research supply group.
Ordered from the loop. Nikon using Kodak Elite II 400 slide film
 Photos were taken with an N70 35mm camera.

A. Pilot studies in guinea pigs Stock trypan blue in saline, PBS, 200 μg / ml or 2 mg / ml gag D
Mixing with NA 1: 1 or with a 77% ratio of trypan blue to DNA gave a final concentration of trypan blue and gag DNA of 5 mg / ml. DNA / trypan blue according to the manufacturer's instructions in No. 2, the weakest permeable syringe tip, while tapping hard to remove any air bubbles that may form between the plunger surface and the o-ring seal. Was loaded to 200 μl. BIOJECTOR fresh or well-filled CO ₂
(Trade name) The syringe tip was previously loaded in an apparatus and fixed in place.

Guinea pigs were anesthetized with a mixture of ketamine (44 mg / kg) and xylazine (5 mg / kg) and the area of the upper hamstring was shaved. A 200 μl injection was then injected into the flesh of the upper hamstring of each guinea pig by using a flat plastic ruler underneath the muscle tissue to support it at the time of injection. The exact site of injection was determined from the anatomy of each animal. The bone extending from the knee to the spine was palpated. To aid in the alignment of the BIOJECTOR ™ syringe tip, a marker was marked 4-6 mm posterior at 2/3 of the knee to spine distance. According to the manufacturer's instructions, stable pressure was absolutely necessary to ensure good contact of the skin retaining ring of the BIOJECTOR ™ syringe tip resulting in good deep intramuscular injection . Without it, the injection would escape from the loosely pressed syringe tip to the side of the syringe without penetrating into the skin and muscle.

After injection of the dye (often done at different DNA concentrations and thus different viscosities of the injection), the guinea pig was sacrificed, closed skin, open skin, muscle cross-section and injection A picture of the object reservoir was taken.

In another experiment, immunization of awake guinea pigs without anesthesia was attempted.
This requires a larger fixed hand (ha) to restrain the guinea pig on the table.
nds) I needed a set. In general, the inventors have found it convenient to support the guinea pig in a sideways position on a 1-2 inch wood block (approximately 6 inches wide and 10 inches long). This allowed the guinea pig to lie directly above the platform surface and allowed better access to restrain the animal with gloved hands. It has also been found that during restraint it is better to calm the animals and cover their eyes.

After injection with the No. 2 tip at various DNA concentrations, the next larger syringe was used until examination of the muscle tissue was either a good intramuscular injection or too strong to penetrate too much. I decided to move to the number (in this case, No. 3 chip). From this procedure, an appropriate tip for the guinea pig upper hamstrings was selected. Also, 27g 1/2 as a control
A direct comparison to the insulin syringe / needle was made.

After performing some pilot experiments on BIOJECTOR ™ 2000 on guinea pigs, we applied a No. 2 syringe tip to the upper flesh of the hamstring hamstring tendon of the guinea pig. Has come to the conclusion that it is the only option. Furthermore, for consistent and reproducible intramuscular injections, it was necessary to support the guinea pig hind limb with a plastic ruler so that the tissue under the BIOJECTOR ™ syringe tip was directly supported. I found it.

Injection of trypan blue / DNA using a 27 g 1/2 insulin syringe / needle gave a bolus of blue DNA formed in the upper hamstring muscle. When DNA / trypan blue was injected intramuscularly into guinea pigs using a No. 2 syringe tip, the blue DNA solution was found to extend over a much larger area and volume of muscle tissue. Structural surfaces within the tissue, for example in the fascia or myofiber layer, allow the blue DNA solution to spread across the axis of injection over a distance of several centimeters from the axis of injection (often in more than one structural surface) I made it. Therefore, this is interpreted as follows. That is, because the resistance to flow or impedance can be reduced at the structural boundaries within the tissue, this may cause the formulation to spread further and immerse a greater tissue surface area and volume than the "bolus" possible from a syringe / needle combination. It is possible. Further, the No. 3 syringe tip almost comes out from the opposite side of the hind limb. This clearly shows that the syringe tip is too powerful for use in guinea pig hamstrings. Therefore, it was confirmed that only No. 2 chip was effective for use in guinea pigs.

Initial experiments were performed on anesthetized animals until we were able to show that there was no detrimental effect of the BIOJECTOR ™ needleless injection device. The inventors then began to restrain guinea pigs by hand on wooden blocks and immunize them without anesthesia. Another site was identified below the hamstrings near the back of the knee joint, but there was much less muscle tissue beneath the skin surface, so the BIOJECTOR ™ injection was not It may be preferable to use a hamstring.

The biodistribution pattern observed after injection of the DNA with BIOJECTOR® was
It was much wider than with a 27g1 / 2 insulin syringe. Without being bound by theory, it is believed that immersing the larger surface area of muscle and other tissues is important to obtaining a larger, more pronounced immune response. As the jet of DNA penetrates the tissue, it may take a less resistant path in a structural surface, such as the fascia or intermuscular boundary, and proceed transversely or perpendicular to the axis of injection. Can be This is unique to injection injection. This is because the solid wall of a standard metal needle prevents the formation of anything other than a bolus of DNA at the end of the needle.

B. Pilot experiments in rhesus monkeys As in the selection experiment of syringe tips for guinea pig biodistribution,
OJECTOR ™ syringe tips were loaded with DNA / trypan blue combinations at various concentrations and injected into various muscle groups of rhesus monkeys scheduled to be euthanized due to other compelling ethical considerations. . The rhesus monkey was anesthetized with a fixed amount of ketamine (10 mg / kg). The primary concern was intramuscular injection into the deltoid or quadriceps (proximal and distal). Other muscle groups injected include biceps, triceps,
Knee flexors (proximal and distal) and calves were included.

After injection of DNA / trypan blue at 0.5 ml / site, the animals were euthanized (Euthanasi
a) Anesthetized with the solution. Shortly thereafter, the same photographic recording scheme was implemented as outlined in the guinea pig study. Syringe tips range from No.2 to No.7,
DNA concentration was 0-5 mg / ml.

After exhalation, the various sites were opened and recorded on film. No.3
Injection of 5 mg / ml DNA into the deltoid muscle of a rhesus monkey with a syringe tip resulted in a well dispersed pattern of blue pigment. This indicates that the DNA has migrated laterally from the injection site. As we did in guinea pigs above, we find that one smaller syringe tip size does not penetrate well into selected muscle tissue (if penetrating it does penetrate a few millimeters into the muscle mass). Or one larger size penetrated too deeply, cutting the muscle tissue and scattered or hit the other side after impact on the bone.

Examination of the injections into rhesus monkeys showed that the skin was thinner below the extremities of the monkey, and therefore less resistant to needle injection. This underscores the importance of selecting a syringe tip appropriate for a given skin / muscle site in a given animal species.

C. Pilot experiment in rabbits A biodistribution syringe tip selection experiment was performed on the dorsal spinal dorsal muscle of awake rabbits. The anterior (left and right) injection sites were approximately 2-3 cm below the height of the posterior rib structure. The posterior site (left and right) was approximately 2-3 cm above the iliac crest. The rabbits were previously shaved and injected with syringe tips No. 2 to No. 7 at a DNA concentration of 0 (PBS) to 5 mg / ml HIV gag DNA. Unlike before, after injection, each side of the spinous muscle was dissected and then sliced thin to visualize the degree of DNA / trypan blue dye distribution. Rabbit skin was much thicker, even after shaving, and was resistant to needleless injection. When using highly viscous 5.5 mg / ml DNA as a vaccine, it was necessary to use a No. 5 syringe tip to achieve a deep 1-2 cm injection. As before, a less effective one size (No. 4) syringe tip only resulted in inadequate injection (penetration up to 5 mm into the muscle), while a more effective one size (No. 4) The syringe tip of .7) provided an injection trajectory that actually crossed the muscle.

Example 4Immunogenicity studies  A. African Green HA / Georgia Immunogenicity Study Ketamine was used to sleep the animals (10 mg / kg). Blood from A / PR / 8/34
Plasmid V1Jns-HA (PR8) encoding hemagglutinin (HA) 2 mg / mL (in 0.15N saline)
) Stock solution in sterile 0.01 M phosphate buffered saline (pH 7.4) to a final concentration of 10 μg / mL
Diluted. DNA solutions were kept on ice until administration. 3.8-7.1kg weight
Plasmids were inoculated into both quadriceps muscles of a group of four African green monkeys of different genders
Seeded. At each injection site, the total plasmid dose per animal should be 10 μg.
0.5 mL of the inoculum was administered. For animals receiving needle injections, the quadriceps in front of both thighs
The injection was made in the largest part near the point. For animals receiving BIOJECTOR (TM) injections
Injected into the anterior lateral part of each thigh using a No. 3 syringe tip. Injection is open to experiment
It was performed at the beginning and boost immunizations were performed after 6 and 18 weeks. Blood is collected at the beginning of the experiment and
And then at 2-4 week intervals, chicken red with 4 HA units of A / PR / 8/34 virus
Coated on hemagglutination inhibition (HI) using blood cells and on 96-well plates
ELISA using all formalin-inactivated A / PR / 8/34 viruses
4 Assay for antibodies to virus, peroxidase to human IgG
Developed with conjugated antibody.

As shown in FIGS. 4 (A) and 4 (B), three injections of 10 μg of DNA with a needle resulted in HI or E
No significant antibody response by LISA was elicited. However, BIOJECTOR (trade name)
Three of four monkeys that received 10 μg of DNA using HI and ELI after the third dose
Respond to antibodies detectable by SA.

B. Guinea Pig Dose-Response Experiments with HIV gag DNA A cohort of six guinea pigs was anesthetized with a ketamine / rompun combination. At weeks 0, 4, and 8, injection with a No. 2 syringe tip or a 27 g 1/2 insulin syringe / needle combination by BIOJECTOR® gave 200
Cohorts were immunized intramuscularly with μl of the V1Jns-HIV gag DNA vaccine. For animals, 200
HIV-1 gag DNA at a concentration of μg / ml, 1 mg / ml or 5 mg / ml (doses 80 mcg, 40
0 mcg or 2 mg).

At weeks 4, 8, and 13, animals were bled and serum was collected and stored at 4 ° C. until assay. Anti-gag Ab ELISA was measured from the sera and titers were assigned from OD plate readings by endpoint titer (criterion of greater than twice background for seroconversion). Typically, the first dilution was 100: 1, but sometimes as high as 400: 1, and serial dilutions were typically less than 3X or 4X plates. Endpoint titers were assigned when the optical density of the color former (OPD) exceeded 2.5 times the background level of the well. Typically, the background level on the plate is about 0.040 OD, so the endpoint titer threshold was typically chosen at 0.100 OD.

As shown in FIG. 5 (A), guinea pigs immunized with BIOJECTOR (trademark) were 80, 400
Or anti-H as determined by ELISA 4 weeks after a single dose of 2000 mcg of HIV-1 gag DNA.
It showed an increase in IV gag antibody titer. At the lowest dose, 1 in 6 animals after needle injection
Only one animal seroconverted, but after immunization with BIOJECTOR®, four out of five assayed seroconverted. At intermediate doses, only 2 out of 6 seroconverted for needle injection, but 6 for needleless injection.
Six of the animals seroconverted. In addition, the cohort geometric mean titer (GMT) showed a 10-fold increase at the 400 mcg dose. At the highest dose of 2 mg, only 2 out of 6 animals seroconverted in the case of needle immunization, while BIOJECTOR®
In the case of needleless injection, 6 out of 6 seroconverted. Surprisingly and surprisingly, the cohort GMTs differed by a significant 80-fold increase.

FIG. 5 (B) shows the anti-gag antibody response from serology 4 weeks after the second immunization with the same vaccine. In this experiment, all cohorts immunized with BIOJECTOR ™ showed 100% seroconversion, whereas the needle-injected cohort did not. At low doses, BIOJECTOR® produced a 12-fold increase in Ab titer, while at intermediate doses it produced a 144-fold increase. This unusually high increase is slightly exaggerated in that three guinea pigs seem to have no response after immunization with a needle at an intermediate dose, thus reducing the needle GMT and thus the relative GMT. The increase factor increases. Regardless, at the higher dose there is a 38-fold increase from needle to needleless syringe.

Finally, after the BIOJECTOR ™ needle-free injection, the insulin syringe /
It should be noted that the variability in the animal's response to the vaccine appears to be surprisingly reduced compared to that seen with the needle. In this experiment, BI
Although all three cohorts injected with OJECTOR® were spread over three well dilutions on the microtiter plate, the syringe / needle group was typically 5 μL on a given plate. Spread over four of the wells.

C. Guinea pig HIV gag DNA anesthesia / site experiment: In another experiment, 8 cohorts of 5 guinea pigs each received 0 and 4
Weekly, again with a No. 2 syringe tip injection with BIOJECTOR ™ or 27 g1 /
Immunization was performed intramuscularly with 200 μl of the vaccine by a two insulin syringe / needle combination. In addition, the animals were injected into the upper hamstring where the large muscle mass is located or into the lower hamstring near the knee joint previously immunized with a conventional needle in our group. In an attempt to test the effects of anesthesia, half of the animals remained awake,
Half were injected under anesthesia (as described above). PB for all guinea pigs
A 1 mg / ml solution of HIV gag DNA in S (dose = 400 mcg) was administered.

At weeks 3 and 7, animals were bled and serum was collected and stored at 4 ° C. until assay. Anti-gag Ab ELISA was measured from the sera and titers were assigned from OD plate readings by endpoint titer (criterion of greater than twice background for seroconversion). Typically, the first dilution was 100: 1, but sometimes as high as 400: 1, and serial dilutions were typically less than 3X or 4X plates. Endpoint titers were assigned when the optical density of the color former (OPD) exceeded 2.5 times the background level of the well. Typically, the background level on the plate is about 0.040 OD, so the endpoint titer threshold was typically chosen at 0.100 OD.

As shown in FIG. 6, the data does not show a significant difference with or without anesthesia.
When comparing the needle to BIOJECTOR®, the increase is 3- to 8-fold. Although the results in guinea pigs with N = 5 per cohort are not very convincing, as before, the BIOJECTOR ™ cohort has improved serum conversion and animal-to-animal compared to syringe / needle. This shows a reduction in variation. It should be noted that this serology is three weeks after the first dose, which is one week earlier than the results described in FIG. 5A.

D. Rhesus monkey HIV gag DNA immunogenicity experiments Recently, immunogenicity studies have been initiated to observe both humoral and cellular immune responses with all HIV gag DNA in non-human primates. Six cohorts of three rhesus monkeys were immunized according to the following study protocol. No. 3 for each animal
Both doses were administered by syringe tip or BIOJECTOR ™ with a 25 g 5/8 needle. A dose of 10 mg / kg ketamine was used as an anesthetic.

Cohort 1 received a total dose of 5 mg DNA under anesthesia with a 25 g 5/8 needle. Cohort 2 has a total dose of BIOJECTOR ™ with No. 3 syringe tip
5 mg of DNA was administered under anesthesia. Cohort 3 was administered under anesthesia with a total dose of 5 mg of the HIV-1 gag DNA construct lacking the leader sequence (DNA # 80) in a BIOJECTOR ™ equipped with a No. 3 syringe tip. Cohort 4 received a total dose of 1 mg DNA under anesthesia with a 25 g 5/8 needle. Cohort 5 has a total dose of BIOJECTOR ™ with No. 3 syringe tip
1 mg of DNA was administered under anesthesia. Cohort 6 has a total dose of BIOJECTOR ™ with No. 3 syringe tip
1 mg of DNA was administered when awake (temporarily restrained with a collar during chair training).

Animals should be bled every other week. Serum will be assayed for anti-gag Ab by ELISA and the cellular response will be characterized by limiting dilution analysis of a CTL-based assay.

Example 5Gene expression research  A. Injected thermostable human placental secreted alkaline phosphatase (SeAP) gene
Lumot 200 encoding the thermostable human placental secreted alkaline phosphatase (SeAP) gene
μl dose of plasmid into BIOJECTOR with 27g1 / 2 needle or No. 2 syringe tip
(Trade name) Needle-free injection device, hemp in four cohorts of eight guinea pigs each
One injection with or without drunk. A small amount of blood over the first two weeks
, And then collected regularly at weeks 3, 4 and 8. Centrifuge blood, remove serum
, Stored at -20 ° C until assay.

FIG. 7 shows SeAP measured in guinea pigs injected with a single injection of high or low doses of SeAP-encoding plasmid DNA by an insulin syringe or BIOJECTOR®.
The results of the activity of the reporter gene are shown. Cohort means are plotted as a function of time. As indicated by the closed circles and squares, SeAP levels in both needle cohorts started at lower levels and increased over the first week. On the other hand, the cohort injected with BIOJECTOR ™ did not show a large response (black diamond). A cohort of high-dose BIOJECTOR (TM)
It showed a 3-fold increase in activity, but declined regularly over the first week. This dramatically different kinetics of gene expression between needle-free injection and syringe / needle injection was neither anticipated nor disclosed in the prior art and was a surprising finding. further,
The ability to take advantage of the apparently rapid increase in gene expression in a short period of time proves to be useful for certain other gene delivery and gene therapy applications (eg, delivery of hormones, receptor proteins and ligands). Maybe.

Without wishing to be bound by theory, it is not intended that the gene or antigen be linked to a short-term inflammatory or immune response that would lead to a large increase in anti-gag Ab endpoint titers using ELISA. It appears that there may be synchronization between the faster kinetics of expression.

Example 6 The size of a needle-free injection tip is determined by US Pat. No. 5,52, incorporated herein by reference.
It is adopted from 0,639.

[0096]

[Table 1]

[Brief description of the drawings]

FIG. 1 shows the results of gel quantification of components of supercoiled DNA (SC) versus total DNA (SC + OC) after various treatments with a needle or needle-free injector. See Example 2B for details.

FIG. 2A shows a gel. In this case, a sample of two different concentrations, slowly dropwise by hand, or by injection by ejection of CO ₂ gas from Biojector (trade name), No.2 (orifice smaller diameter) or No.5 (large Diameter orifice) BI
Passed through an OJECTOR® syringe tip. See Example 2C for details.

FIG. 2B shows the results of gel quantification. In this case, two different concentrations of the sample are
No. 2 (small diameter orifice) or No. 5 (large diameter orifice) BIOJECTOR (trademark) syringe tip, slowly and drop by hand or by jetting with CO ₂ gas from BIOJECTOR Passed through. See Example 2C for details.

FIG. 3A shows a gel. In this case, a sample of two different concentrations, slowly dropwise by hand, or by injection by ejection of CO ₂ gas from Biojector (trade name), No.2 (orifice smaller diameter) or No.5 (large Diameter orifice) BI
Passed through an OJECTOR® syringe tip. See Example 2D for details.

FIG. 3B shows the results of gel quantification. In this case, two different concentrations of the sample are
No. 2 (small diameter orifice) or No. 5 (large diameter orifice) BIOJECTOR (trademark) syringe tip, slowly and drop by hand or by jetting with CO ₂ gas from BIOJECTOR Passed through. See Example 2D for details.

FIG. 4 (A) shows hemagglutination inhibition of a group of four African green monkeys immunized three times with 10 μg of influenza HA DNA per immunization by needle or BIOJECTOR ™ (immunization).
HI) Indicates response. See Example 4A for details.

FIG. 4 (B) shows 10 μg of HA DN per immunization by needle or BIOJECTOR (trade name).
2 shows the ELISA antibody response of a group of four African green monkeys immunized three times with A. See Example 4A for details.

FIG. 5 (A) shows B, 4 weeks after administration of a single dose of 80, 400 or 2000 mcg of HIV-1 gag DNA.
Figure 3 shows increased anti-HIV-1 gag antibody titers (measured by ELISA) in guinea pigs immunized with IOJECTOR® as compared to those obtained with needles. Details are examples
See 4B.

FIG. 5 (B) shows the anti-gag antibody response 4 weeks after the second immunization with the same vaccine at 4 weeks. See Example 4B for details.

FIG. 6 shows a cohort of five guinea pigs after immunization of the upper or lower hamstring site using a needle or BIOJECTOR® needleless syringe in the presence or absence of anesthesia. The results are shown. See Example 4C for details.

FIG. 7 shows high or low doses of human thermostable secretory alkaline phosphatase (SeAP) encoding plasmid DNA by insulin syringe or BIOJECTOR ™.
5 shows the results from the reporter gene activity of SeAP measured in guinea pigs injected once. Cohort means are plotted as a function of time. See Example 5A for details.

FIG. 8A shows reporter gene activity of human thermostable secreted alkaline phosphatase (SeAP) measured in guinea pigs injected once with high or low doses of SeAP-encoding plasmid DNA by an insulin syringe or Biojector ™. The results from are shown. Individual animal values are plotted as a function of time. Day 2 is shown in FIG. 8A. See Example 5A for details.

FIG. 8B. Reporter gene activity of human thermostable secreted alkaline phosphatase (SeAP) measured in guinea pigs injected once with high or low doses of SeAP-encoding plasmid DNA by insulin syringe or Biojector ™. The results from are shown. Individual animal values are plotted as a function of time. Day 4 is shown in FIG. 8B. See Example 5A for details.

FIG. 8C. Reporter gene activity of human thermostable secreted alkaline phosphatase (SeAP) measured in guinea pigs injected once with high or low doses of SeAP-encoding plasmid DNA by insulin syringe or Biojector ™. The results from are shown. Individual animal values are plotted as a function of time. Day 7 Figure 8
Shown in C. See Example 5A for details.

FIG. 9 shows the results in saline or saline / glycerol at various temperatures and DNA concentrations.
4 shows the results from a viscosity measurement of DNA. See Example 2E for details.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 5/00 B 15/09 15/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, (KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AU, AZ, BA, BB, BG, BR, BY, CA, CN, CU, CZ, EE, GD, GE, HR, H U, ID, IL, IN, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LV, MD, MG, MK, MN, MX, NO, NZ, PL, RO, RU, SG , SI, SK, SL, TJ, TM, TR, TT, UA, US, UZ, VN, YU, ZA. Avenue 126 (72) Inventor Bolkin, David United States, New Jersey 07065, Lowway, East Lincoln Avenue 126 (72) Inventor Simon, Adam United States, New Jersey 07065, Lowway, East Linker Avenue 126 F term (reference) 4B024 AA01 CA04 EA02 FA02 GA11 HA17 4B065 AA90X AA95Y AB01 CA45 4C076 AA12 BB11 CC06 DD23D DD26Z 4C 084 AA13 MA66 NA10 ZB092 ZC552

Claims (9)

[Claims] 1. An improved method of inducing an immune response to a polynucleotide vaccine in an animal using a needle-free injector, comprising introducing the vaccine intramuscularly, wherein the polynucleotide vaccine comprises A method wherein the polynucleotide vaccine is not present in the bolus upon entry into the animal, and the polynucleotide vaccine is substantially uniformly distributed within the area of muscle tissue. 2. The polynucleotide vaccine according to claim 1, wherein said polynucleotide vaccine is a DNA vaccine.
The method described in. 3. The polynucleotide vaccine of claim 2, wherein said polynucleotide vaccine is an HIV vaccine.
The method described in. 4. The polynucleotide vaccine encodes HIV gag.
The method according to claim 3. 5. The method of claim 2, wherein said polynucleotide vaccine encodes influenza HA. 6. The method of claim 1, wherein introduction of said vaccine is controlled by passing said vaccine through an orifice having a predetermined diameter. 7. The method of claim 1, wherein the concentration of said polynucleotide vaccine is at least 5 μg / ml. 8. A method of delivering DNA to an animal for gene therapy using a needle-free injector, comprising introducing the vaccine intramuscularly, wherein the polynucleotide vaccine comprises the animal. Upon entry into the bolus, and wherein the polynucleotide vaccine is substantially uniformly distributed within the area of muscle tissue upon entry into the animal. 9. The method of claim 8, wherein said polynucleotide vaccine is in an amount sufficient to produce a desired prophylactic or therapeutic response.