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DNA vaccine formulations

a technology of nucleic acid and vaccine, applied in the field of new formulations of nucleic acid pharmaceutical products, can solve the problems of significant enhancement of stability, no increase in stability, dna degradation, etc., and achieve the effects of improving stability, improving stability, and increasing dna stability

Inactive Publication Date: 2002-10-24
MERCK & CO INC
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Problems solved by technology

In addition, our data indicates that specific chelating agents such as inositol hexaphosphate, tripolyphosphate, succinic and malic acid, increase the stability of plasmid DNA in storage, while other commonly used chelating agents such as EDTA, desferal, ethylenediamine-Di(o-hydoxy--phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA) provide no significant enhancement of stability.
Our results also indicate that several scavengers expected to stabilize DNA (based on known rate constants with hydroxyl radical) unexpectedly accelerated DNA degradation, or provided no increase in stability.
Influenza can cause significant systemic symptoms, severe illness (such as viral pneumonia) requiring hospitalization, and complications such as secondary bacterial pneumonia.
Development of the next season's vaccine relies upon predicting the upcoming circulating strains (via sentinel sampling in Asia), which is inexact and can result in a poor match between strains used for the vaccine and those that actually circulate in the field.
However, due to the length of time needed to make and formulate the current licensed vaccine, the new vaccine strain could not be introduced during the 1992-1993 season despite the evidence for poor protection from the existing vaccine and the increased virulence of the new circulating H3N2 strain.
Characteristics of an Ideal Universal Influenza Vaccine include the following:
1) Generation of group-common (heterologous) protection.
2) Increased breadth of antibody response. Because CTL are thought to play a role in recovery from disease, a vaccine based solely upon a CTL response would be expected to shorten the duration of illness (potentially to the point of rendering illness subclinical), but it would not prevent illness completely.
3) Increased duration of antibody responses. Because one of the very groups that is at highest risk for the morbidity and mortality of influenza infection (elderly) is also the group in whom protective antibody titers may decline too rapidly for annual immunization to be effective, an improved vaccine should generate protective titers of antibody that persist longer.
In addition, our data indicates that specific chelating agents such as inositol hexaphosphate, tripolyphosphate, succinic and malic acid, increase the stability of plasmid DNA in storage, while other commonly used chelating agents such as EDTA, desferal, ethylenediamine-Di(o-hydoxy--phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA) provide no significant enhancement of stability.
Our results also indicate that several scavengers expected to stabilize DNA (based on known rate constants with hydroxyl radical) unexpectedly accelerated DNA degradation, or provided no increase in stability.
In addition, our data indicates that specific chelating agents such as inositol hexaphosphate, tripolyphosphate, succinic and malic acid, increase the stability of plasmid DNA in storage, while other commonly used chelating agents such as EDTA, desferal, ethylenediamine-Di(o-hydoxy--phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA) provide no significant enhancement of stability.
Our results also indicate that several scavengers expected to stabilize DNA (based on known rate constants with hydroxyl radical) unexpectedly accelerated DNA degradation, or provided no increase in stability.
Moreover, the results suggest that EDTA alone decreases DNA stability in the absence of ethanol, but increases DNA stability in the presence of ethanol.

Method used

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Examples

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example 2

[0089] Influenza Virus Gene Constructs In Expression Vector V1j

[0090] Many of the genes from the A / PR / 8 / 34 strain of influenza virus were cloned into the expression vector V1J, which gives rise to expression at levels as high or higher than in the V1 vector. The PR8 gene sequences are known and available in the GENBANK database. For each of the genes cloned below, the size of the fragment cloned was checked by sizing gel, and the GENBANK accession number against which partial sequence was compared are provided. For a method of obtaining these genes from virus strains, for example from virus obtained from the ATCC (A / PR / 8 / 34 is ATCC VR-95; many other strains are also on deposit with the ATCC).

[0091] A. Subcloning the PR8 Genes into V1J:

[0092] 1. NP

[0093] The NP gene was subcloned from pAPR501 (J. F. Young, U. Desselberber, P. Graves, P. Palese, A. Shatzman, and M. Rosenberg (1983), in The Origins of Pandemic Influenza Viruses, ed. W. G. Layer, (Elsevier, Amsterdam) pp.129-138). It wa...

example 3

[0110] Growth of Microbial Cells, Cell Lysis and Clarification

[0111] One liter of frozen E. coli cell slurry was used to make 8 liters of cell suspension in STET buffer (8% sucrose, 0.5% TRITON, 50 mM TRIS buffer, pH 8.5 and 50 mM EDTA). The absorbance of the cell suspension at 600 nm was about O.D. 30. The suspension was stirred continuously to ensure homogeneity. The viscosity of the cell suspension was measured to be about 1.94 cp at room temperature (24.degree. C.). The cell suspension was pumped through the heat exchanger at 81 mL / min which corresponded to a residence time of the cell solution in the heat exchanger of about 35 seconds. The bath temperature was maintained at 92.degree. C. The inlet and outlet temperatures of the cell solution were measured to be about 24.degree. C. and about 89.degree. C. (average), respectively. About 1 liter of sample was run through the heat exchanger. No visible clogging of the tube was observed although the lysate was somewhat thicker than ...

example 4

[0113] Control and Reproducibility of Cell Lysis with the Heat Exchanger

[0114] Adjusting the flowrate (i.e., residence time) at which the cell slurry is pumped through the heat exchanger permits tight control of the temperature of lysis, i.e., the outlet temperature. A cell slurry solution was prepared as described in Example 3 and pumped through the heat exchanger at flow rates ranging from 160 to 850 mL / min. The corresponding outlet temperatures ranged between 93.degree. C. and 65.degree. C., respectively. The initial temperature of the cell slurry was 24.degree. C. and the bath temperature was kept constant at 96.degree. C. In addition, a number of runs were performed where an outlet temperature of 80.degree. C. was targeted. Yields of 24 mg of circular DNA per L of clarified supernatant were consistently obtained demonstrating the reproducibility of the process.

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Abstract

This invention relates to novel methods and formulations of nucleic acid pharmaceutical products, specifically formulations of nucleic acid vaccine products and nucleic acid gene therapy products. The formulations of the disclosure stabilize the conformation of DNA pharmaceutical products.

Description

BACKGROUND OF THE DISCLOSURE[0001] This invention relates to novel formulations of nucleic acid pharmaceutical products, specifically formulations of nucleic acid vaccine products and nucleic acid gene therapy products. The formulations of the disclosure stabilize the conformation of DNA pharmaceutical products. The vaccines, when introduced directly into muscle cells, induce the production of immune responses which specifically recognize human influenza virus.[0002] During storage as a pharmaceutical entity, DNA plasmid vaccines undergo a physicochemical change in which the supercoiled plasmid converts to the open circular and linear form. A variety of storage conditions (low pH, high temperature, low ionic strength) can accelerate this process. In this invention, the removal and / or chelation of trace metal ions (with succinic or malic acid, or with chelators containing multiple phosphate ligands) from the DNA plasmid solution, from the formulation buffers or from the vials and clo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61KA61K39/145A61K47/02A61K47/10A61K47/12A61K47/18A61K47/20A61K48/00
CPCA61K9/0019A61K39/145A61K47/02A61K47/10A61K47/12C12N2760/16134A61K47/20A61K48/00A61K2039/53C12N15/1003C12N2760/16111A61K47/183A61K39/12
Inventor VOLKIN, DAVID B.EVANS, ROBERT K.BRUNER, MARK
Owner MERCK & CO INC
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