Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
2001-05-14
2003-09-16
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S320100, C536S024100
Reexamination Certificate
active
06620595
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to retroviral vectors. The invention particularly relates to retroviral vectors that have an enhanced 3′ transcription termination structure and to methods for using such vectors to express heterologous coding sequences in mammalian cells and organisms.
BACKGROUND OF THE INVENTION
Retroviral Vectors
Retroviral vectors are currently one the most frequently used gene delivery vehicles in gene therapy protocols. Fundamental to the utility of retroviral vectors is the various retrovirus characteristics retained by the vectors. Such characteristics include efficient transfection of many cell types and stable integration of their genomes into a host cell chromosome, which enables long-term expression of vector encoded genes. Another important retained characteristic is that the initial steps of the vector life cycle from binding of vector particles through integration of its genome into a host cell's genetic material require no de novo synthesis of viral proteins.
Basic Components of Retroviral Vectors
The main features of the wild-type retroviral genome are summarized in
FIG. 1
, which shows the open reading frames and the structures of the viral long terminal repeats (LTRs). Retroviral vectors comprise genomes derived from retroviruses. The simplest type of retroviral vectors have a significantly pared down retroviral genome which is missing most of the sequences encoding viral genes (e.g., gag, env and pol) and retains only sequences that are required for the packaging, reverse transcription and integration. The pared down retroviral genomes are often referred to as retroviral backbones, upon which further modifications can be made and to which heterologous genes and sequences can be added to form retroviral vectors. Typically, a heterologous gene is inserted into the backbone in such a way that allows the 5′ LTR promoter to drive its subsequent expression. An expression construct comprising a heterologous gene operatively associated with a promoter can also be inserted into the backbone for delivery and expression in a target cell.
Retroviral vectors missing some or all of the viral genes are replication deficient. Production of viral particles comprising such vectors requires vector propagation in host cells that provide the missing functions in trans. Trans complementation can be achieved in various ways including transfecting the host cell with a packaging helper construct, also derived from a retroviral genome, which expresses the missing viral proteins but cannot be packaged because of a deletion of the packaging signal. This system of retroviral vector production is illustrated in FIG.
2
. When both the vector and packaging helper construct are present in a producer cell, infectious retroviral particles are released that are capable of delivering the vector genome with its inserted gene. This process of gene delivery is referred to as transduction.
Lentiviral Vectors
To date, the most common retroviral vectors used in clinical gene therapy protocols have been based on the murine leukemia virus (MuLV), and a variety of packaging systems to enclose the vector genome within viral particles have been developed (reviewed in Miller, A D. 1997. Development and applications of retroviral vectors. In Retroviruses, Ed. Coffin J M, Hughes S H, Varmus H E. CSHL Press, New York.). The vectors themselves have all of the viral genes removed, are completely replication-defective, and can accept up to approximately 6-8 kb of exogenous DNA. These current vector/packaging systems seem to pose minimal risk to patients, and to date there have been no reports of toxicity or long-term problems associated with their use.
However, MuLV and vectors derived from it are only able to infect dividing cells. This is because the pre-integration nucleoprotein complex is unable to cross an intact nuclear membrane. In contrast, the prototypical lentivirus HIV-1 has been shown capable of nuclear import even when an intact membrane exists, and HIV-1-derived vectors are therefore able to transduce non-dividing cells (Naldini et al., Science 272:263-267 (1996)). This property of HIV vectors makes them particularly attractive candidates for gene therapy when the target cell is non-dividing and stable integration of the heterologous gene is required.
Improvements in Retroviral Vector Design and Production Systems
The retroviral vector production system described above is functional but unsatisfactory in several ways. In particular, overlaps that remain between the vector sequences and sequences encoding viral components in packaging helper constructs means that there is a significant risk of recombination events that would create an infectious replication-competent retrovirus (RCR). Such overlaps exist largely because extensive sequences of the gag gene are retained in the vector to enhance packaging efficiency. In addition, the LTRs are frequently retained in packaging helper constructs to provide both promoter and polyadenylation sequences.
In order to minimize the risk of RCR production, various improved approaches to vector design and production have been developed. One approach splits the packaging components, placing the gag-pol genes and the env gene onto separate plasmids that can be individually introduced into the packaging cell. In another approach, Env-mediated recombination is avoided by the use of heterologous envelope proteins whose coding sequences have no homology with the genome of the parental retrovirus but which can be incorporated into the vector particle (a process referred to as pseudotyping). A commonly used heterologous envelope protein is VSV G, the G protein from vesicular stomatitis virus (Burns et al., Proc. Natl. Acad. Sci. 90:8033-8037 (1993)). See
FIG. 3
, Panel A.
In yet another approach, LTR-mediated recombination is reduced by the use of heterologous promoters and polyadenylation signals in the packaging helper constructs. This can also have the advantage of enhancing vector titer (Soneoka et al., Nucleic Acids Res 25:628-633 (1995)). This approach typically involves deleting non-essential sequences from the vector LTRs and where appropriate, replacing the deleted sequences with heterologous sequences. For example, heterologous promoters, such as the CMV immediate-early promoter, have been used to replace the 5′ U3 promoter. In other instances, 3′ U3 sequences have been significantly deleted, as is the case with self-inactivating (SIN) vectors, as long as the integrase recognition sequences (i.e., att sequences) are retained (Yu et al., Proc. Natl. Acad. Sci 83:3194-3198 (1986)). See
FIG. 3.
, Panel B.
These approaches have been used in developing various lentivirus-based vectors, which raise special safety concerns because of the possibility of pathogenic RCR arising from recombination events. Example products of this approach include the CMV-driven SIN vectors (Zufferey et al., J. Virol. 72:9873-9880 (1988)), the minimal packaging helper constructs with all of the non-essential genes inactivated or removed (Zufferey et al., Nat. Biotechnol. 15:871-875 (1997)), and retroviral particles comprising non-HIV-1 envelope proteins such as the VSV G.
Retroviral Vector Integration
Retroviral vectors integrate their genomes into a host cell's genetic material. A great deal is known about the process of retroviral integration, which is carried out by the viral integrase. Integrase recognizes sequences at the ends of the LTRs of the DNA provirus (the att sites, FIG. Panel
1
B), and inserts the provirus more or less randomly into the host genome, although some sequence preferences have been reported (Carteau et al., J. Virol. 72:4005-4014 (1988)).
The ability of retroviral vectors to integrate is a two-edged sword. On the one hand, it allows for the possibility of stable long-term expression of vector encoded genes, with the integrated provirus being passed on to all daughter cells. On the other hand, vector integration can interfere with the normal functioning of flanking host genes. Indeed, retro
Barcova Maria
Cannon Paula Marie
Golightly Douglas A.
Jen George C.
Ketter James
Meigs J. Timothy
Sullivan Daniel M.
LandOfFree
Retroviral vectors comprising an enhanced 3′... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Retroviral vectors comprising an enhanced 3′..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Retroviral vectors comprising an enhanced 3′... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3060237