Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-08-07
2001-12-11
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S235100, C435S320100, C435S069100, C435S455000, C435S456000, C435S457000, C435S325000, C435S366000, C435S091400, C536S023100, C536S023720, C536S024100, C536S024200
Reexamination Certificate
active
06329181
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention provides methods, host cells, and vectors which permit efficient production of recombinant parvovirus virions. In particular, the present invention relates to parvovirus helper functions that provide for high-efficiency recombinant parvovirus production but reduce the potential of generating replication competent particles.
Parvoviruses vectors, such as adeno-associated virus (AAV) vectors are useful for gene therapy. In general, recombinant adeno-associated virus (rAAV) vectors are generated by transfection of an AAV vector plasmid and a helper plasmid in the presence of helper virus infection (Samulski, et al. (1989)
J Virol
63: 3822-3828). The AAV vector is constructed by replacing the whole coding region of the AAV genome with a transgene. This creates a defective AAV vector which is incapable of replication. In order to provide the necessary helper functions, a helper plasmid can be constructed. The helper plasmid contains the AAV Cap and/or Rep coding region, but lacks the AAV inverted terminal repeat sequences. Accordingly, the helper plasmid can neither replicate nor package itself. After the AAV helper plasmid and the AAV vector are introduced into a host cell, the transfected cells can be infected with a helper virus, for example, an adenovirus, which, among other functions, transactivates the AAV promoters present on the helper plasmid that direct the transcription and translation of AAV Rep and Cap regions. Upon subsequent culture of the host cells, recombinant AAV virions (harboring the transgene) are produced.
Although there is no overlapping sequence between the AAV vector and the helper plasmid, the probability of generating replication competent AAV (rcAAV) particles through non-homologous recombination, is relatively high (Allen et al. (1997)
J Virol
71: 6816-6822). These replication competent particles affect transgene expression (Grimm, et al. (1999)
Hum Gene Ther
10: 2445-2450), are a safety hazard in applications of AAV vectors for human gene therapy, and also reduce the yield of recombinant AAV virions.
Previous attempts to address the problem of rcAAV particles includes using heterologous promoters for driving the Rep coding and Cap region, separating the Cap and Rep coding regions into different vectors (See Allen, et al. (1997) J Virol 71: 6816-6822 and Flotte, et al. (1995)
Gene Ther
2: 29-37), and using truncated AAV terminal repeat sequences (Wang, et al. (1998)
J Virol
72: 5472-5480). Although these approaches reduced the number of replication competent particles, the replication competent particles were still present in large scale preparations. Accordingly, a need exists for methods and compositions of producing recombinant viral vectors without the presence for contaminating replication competent particles. A need also exists for methods of producing recombinant AAV virions without the presence of contaminating replication competent particles.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that recombinant parvovirus virions can be produced at a higher titer, and without detectable quantities of replication competent particles, using the helper functions of the invention. The helper functions comprise at least one intron sequence inserted at one or more positions within a non-structural coding region, and/or a structural coding region of a parvovirus genome. The intron sequences can be non-native intron sequences, which are not typically present in a parvovirus genome, for example, a &bgr;-globin intron sequence. The intron sequence can be a native intron sequence that is typically present in a parvovirus. At least one native intron sequence can be inserted at one or more positions within the non-structural coding region and/or the structural coding region of a parvovirus genome. The invention also encompasses inserting a combination of a native intron sequence and a non-native intron sequence in at least one or more positions within the non-structural coding region and/or the structural coding region of a parvovirus genome. The technology described herein enables the rapid and efficient generation of recombinant parvovirus virions with a reduced titer, or without the presence of detectable replication competent particles. In particular, the invention provides nucleic acid molecules that encode parvovirus helper functions containing at least one native and/or non-native intron sequence, and methods for producing recombinant parvovirus virions using such helper functions.
The intron sequence can be inserted into one or more positions in a non-structural protein coding region, for example, the Rep coding region. The intron sequence can be inserted into one or more positions in a structural protein coding region, for example, the Cap coding region, or any combination thereof. Introduction of at least one intron sequence to the structural and/or non-structural protein coding regions reduces and/or eliminates the number of contaminating replication competent particles generated during recombinant viron production.
Accordingly, in one aspect, the invention features a nucleic acid molecule encoding a parvovirus helper function. The nucleic acid molecule comprises a non-structural protein coding region derived from a parvovirus, a structural protein coding region derived from a parvovirus, and at least one intron sequence inserted at one or more positions within said regions, such that the intron sequence increases the size of the nucleic acid molecule to a size larger than a nucleic acid molecule without the intron sequence, wherein the increase in size prevents packaging of a pseudo wild-type parvovirus into a replication competent particle.
In one embodiment, the nucleic acid molecule encoding a parvovirus helper function is an adeno-associated virus selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5 and AAV-6, preferably, AAV-2. The non-structural protein can be a protein such as a Rep protein and the structural protein can be a Cap protein. Intron sequences are inserted to increase the size of the viral genome. In one embodiment, the intron sequence can be at least one non-native intron sequence i.e., an intron sequence that is not typically found in the viral genome. Examples of non-native sequences include, but are not limited to, &agr;-globulin intron, &bgr;-globulin intron, collagen intron, ovalbumin intron, SV40 intron and p53 intron. The non-native intron sequence can also be derived from an autonomous parvovirus such as, LUIII, minute virus of mice (MVM), human parvovirus B19, hamster parvovirus, feline panleukopenia virus, canine parvovirus porcine parvovirus, latent rat parvovirus and mink enteris parvovirus. In another embodiment, the intron sequence can be a native intron sequence, which is typically present in the parvoviral genome, i.e., at least one intron sequence that is typically found in the parvovirus.
A particularly useful parvovirus is AAV-2, accordingly the invention features helper functions that contain nucleic acids encoding the Rep and/or the Cap proteins for AAV-2 with at least one intron sequence. In a helper function with a nucleic acid encoding both the Cap and the Rep proteins, at least one intron sequence can be inserted into the nucleic acid encoding the Cap coding region, the Rep coding region, or both the Cap coding region and the Rep coding regions.
Accordingly, in one aspect, the invention features a nucleic acid molecule encoding an adeno-associated virus (AAV) helper function. The nucleic acid comprises a Rep coding region derived from an AAV, a Cap coding region derived from an AAV, and at least one intron sequence inserted at one or more positions within the Cap coding region and the Rep coding region, such that the intron sequence increases the size of the nucleic acid molecule to a size larger than a nucleic acid molecule without the intron sequence, wherein the increase in size prevents packaging of a pseudo wild-type AAV into a replication competent particle.
The invention also features a helper f
Cao Lei
During Matthew J.
Xiao Weidong
Engellenner Thomas J.
Guzo David
Neurologix, Inc.
Nutter & McClennen & Fish LLP
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