Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-02-16
2003-03-25
Ketter, James (Department: 1633)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S468000, C435S472000, C435S320100, C435S235100, C424S450000, C424S486000
Reexamination Certificate
active
06537813
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to concurrent flow mixing methods and apparatuses that are adapted for preparation of gene therapeutics, as well as compositions prepared thereby. In the various embodiments, the present invention provides controlled and uniform mixing of gene therapy vectors and gene therapy vector vehicles for improved reproducibility, scaleability, stability, and pharmaceutical efficacy.
BACKGROUND
Ideally, techniques for nucleic acid delivery include one or more attributes, such as, for example: 1) efficient preparation of the nucleic acid delivery composition, including simplified operation, cost effectiveness, consistency, stability and uniformity; 2) efficient delivery and incorporation of the nucleic acid into the host cell; 3) avoidance of undesirable side effects such as cell toxicity, or the introduction of unwanted elements (e.g., additional viral genes); and 4) selective targeting of the nucleic acid to the desired host cell.
Various methods have been employed to introduce foreign genes into cells. Since DNA is a large and bulky molecule, it is no simple matter to introduce such molecules into cells, not only due to the size of the molecule but due to its chemical and charge-related characteristics. Therefore, many gene therapy methods endeavor to find a way to “package” the therapeutic DNA in such a manner that it may be transported across the membranes of the target cells. As those of skill in the art are well aware, this is not an easily-achieved goal. Moreover, most “packages” are comprised of components which interact with each other based on cooperative kinetics. Such kinetics are associated with bringing together components that each contain a variety of cooperative binding sites, thereby adding a level of complexity which when poorly controlled may lead to non-uniform compositions. Accordingly, to date, no methods are known to have been developed which control the cooperative kinetics involved in the manufacture of gene therapeutic compositions nor have methods been developed which allow for consistent, reproducible, and uniform (i.e., substantially homogenous) gene therapeutic compositions to be made.
One method of introducing therapeutic nucleic acids into host cells utilizes polycation
ucleic acid condensates. See, for example U.S. Pat. Nos. 5,166,320 and 5,635,383 as well as published International Patent App. Nos. WO 93/04701 and WO 94/06922. This methodology uses polycations to condense the nucleic acid into a compact structure so as to facilitate cellular uptake. However, such methodologies suffer from poorly controlled conditions for forming the condensates, thereby leading to aggregation or poorly condensed compositions.
Another current method of introducing foreign (or therapeutic) DNA into cells uses precipitation of DNA with calcium phosphate to form insoluble particles. Ideally, these particles become internalized in the host cells (via endocytosis) and induce expression of the new gene. Internalization of such particles, however, is independent of an endocytosis recognition site, so the internalization is non-specific and is thus not targetable to particular cells and organs. While generally applicable to in vitro applications, this process has more limited applicability in in vivo applications due to the insolubility of the DNA co-precipitate particles and the inability to control uptake and targeting specificity. Additionally, conditions necessary for internalization of DNA using the calcium phosphate method may be harsh and cause cell death.
An alternate method of introducing foreign genes into cells utilizes liposomes. Liposome-encapsulated DNA has been utilized both in vitro and in vivo. This technique, however, suffers from difficulties in controlling liposome size, which inherently affects uniform and controlled delivery to the target cell. There are also problems associated with keeping the liposome intact throughout the cell delivery process and difficulties associated with achieving specific targeting.
Other methods of delivering exogenous nucleic acids to cells have been proposed for use but suffer from a variety of limitations that make them impractical for gene therapy applications. For example, electroporation is impractical as the conditions required for gene delivery are harsh and lead to toxicity and cell death. Similarly, injection of naked DNA is unpredictable, especially with respect to targeting specificity, controlled uptake, and immunogenicity. Microinjection of nucleic acids into cells is very labor intensive, because each cell must be individually manipulated, and thus it is not a practical alternative to bolus injections of DNA, although it avoids some of the problems associated with bolus injections. Thus, it is clear that none of the foregoing techniques provide a practical alternative for in vivo therapeutic applications.
The preparation of gene therapy vectors according to the methods described herein thus provides attractive means to improve upon known techniques and develop new gene transfer methods. The preparation and use of such vectors is not without its own unique limitations, however, as many have observed. Fortunately, the methods and apparatuses of the present invention overcome many of the difficulties experienced by others and enhance the efficacy of gene therapy for pharmaceutical commercialization.
Various methods of compacting nucleic acids to facilitate their entry into cells are described in the art and are useful to underscore the novel aspects of some embodiments of the present invention. The following examples are provided to illustrate some of those methods.
International Patent Publication WO 95/25809 describes nucleic acids that are compacted to facilitate their uptake by target cells of an organism. The publication further describes methods for compacting nucleic acids and therapeutic uses of the compacted DNA for delivery across the membrane of living cells.
International Patent Publication WO 96/41606 describes synthetic virus-like particles containing a plurality of peptides capable of condensing nucleic acid and condensed nucleic acid. The synthetic virus-like particle is self-assembling and may be designed to deliver nucleic acid to be incorporated into the chromosomal or extrachromosomal sequences of target cells. These particles are described as being useful for transfecting mammalian cells.
Other systems that have been developed to deliver nucleic acid molecules to mammalian cells include, emulsions (e.g., liposomes), nanoparticles, microparticles, and similar heterogeneous systems. However, these systems also suffer from lack of scaleable production methodologies that produce uniform, reproducible and highly efficacious compositions.
Of the aforementioned methods, compaction or condensation of the nucleic acids for delivery into the host cell offers great potential. For this technique to be fully exploited, however, methods and apparatuses for producing suitable nucleic acid complexes on a variety of scales, including for example, laboratory scale, commercial production scale, and individual patient bedside administration scale, are desirable. While a variety of mixing devices are available, some of which are exemplified below, they are not generally applicable to gene therapy vector and vehicle compositions and especially with regard to nucleic acid compaction or condensation methods.
For example, U.S. Pat. No. 4,908,187 describes a diluting and mixing device which is capable of diluting a first solution to produce a second solution which is mixed with an undiluted third solution to produce a unique series of combined solutions. Such a device is intended for use in obtaining kinetic analysis (e.g., chemical, biochemical or physical chemical) data on reactions in solution.
U.S. Pat. No. 4,979,942 describes a two-component syringe delivery system. In this system, two reactive fluids are delivered simultaneously and separately from a pair of syringes to a delivery site. The tubing exiting one syringe passes through a cannula exiting from the
Chen Xian
D'Andrea Mark J.
Ketter James
Schnizer Richard
Seed Intellectual Property Law Group PLLC
Selective Genetics Inc.
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