Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1994-06-30
2001-03-20
Martinell, James (Department: 1633)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S440000, C514S04400A
Reexamination Certificate
active
06204059
ABSTRACT:
INTRODUCTION
The present invention relates to the production of adeno-associated virus (AAV) capsids in vivo or in vitro which may be used to transfer native or heterologous molecules into appropriate host cells. The invention further relates to the production of recombinant AAV capsids engineered to carry heterologous antigens for the stimulation of an immune response.
BACKGROUND OF THE INVENTION
The current interest in molecular replacement therapy as a modality for clinical treatment has necessitated the development of methods to safely and efficiently deliver genetic material or other molecules to cells. This has been attempted using physical means of cell permeation or by employing biological agents that naturally infect a host cell.
PHYSICAL TRANSFER METHODS
Methods to transfer DNA into a recipient cell include standard transfection techniques, mediated by calcium phosphate or DEAE-dextran, electroporation of accessible cells, and liposome-mediated transfer. These techniques are utilized in the research setting but require in vitro handling of the recipient cells and thus have limited clinical potential.
VIRAL VECTOR SYSTEMS
Viral vector systems exploit the efficiency of natural infection and use of DNA technology to engineer recombinant viruses that carry heterologous genes into the cell. Most clinical trials to date have taken this approach (Morgan, R. A. and W. F. Anderson, Annu. Rev. Biochem. 62:191-217, 1993).
Retroviral vectors have been most commonly used, chiefly because they can facilitate the integration of the carried DNA into the host cell genome, establishing stable integrants which are amplified during cellular DNA replication. However, this capability, while circumventing the limitations of transient gene expression, can result in the inadvertent activation of host genes or the interruption of cellular coding sequences due to random integration.
Adenoviral vectors can introduce DNA into a cell, but do not support the integration of the genetic material, which remains in episomal form in the nucleus and does not co-replicate with the cellular DNA. While adenoviruses are minor pathogens, their optimization as clinically relevant transfer vehicles may be limited to those tissues that are natural hosts for these viruses, i.e., the lungs (Berkner, K., Curr. Topics. Micro. Immunol. 158:39-66, 1992).
There is a clear need for safer delivery systems that combine the efficiency of viral infection with the potential to deliver genetic material to a targeted integration site in the host cell genome. There is also the need to be able to construct molecular delivery vehicles in vitro which can then be packaged in a cell-free system and which are capable of encapsidating a wide range of molecular constituents.
ADENO-ASSOCIATED VIRUS
AAV is a parvovirus that can assume two pathways upon infection of a host cell. In the presence of helper virus, AAV will enter the lytic pathway where the viral genome is transcribed, replicated, and encapsidated into newly formed viral particles. In the absence of helper virus function, the AAV genome becomes integrated as a provirus into a specific region of the host cell genome, through recombination between the AAV termini and host cell sequences. Characterization of the proviral integration site and analysis of flanking cellular sequences indicates specific targeting of AAV viral DNA into the long arm of human chromosome 19 (Kotin, R. M., et al., Proc. Natl. Acad. Sci. USA 87:2211-2215, 1990; Samulski, R. J., et al., EMBO J. 10:3941-3950, 1991). This particular feature of AAV reduces the likelihood of insertional mutagenesis resulting from random integration of viral vector DNA into the coding region of a host gene. Furthermore, in contrast to the retroviral LTR sequences, the AAV ITR sequences appear to be devoid of transcriptional regulatory elements, reducing the risk of insertional activation of protooncogenes.
The AAV genome is composed of a linear single stranded DNA molecule of 4680 nucleotides which contains major open reading frames coding for the Rep (replication) and Cap (capsid) proteins. Flanking the AAV coding regions are two 145 nucleotide inverted termini (ITR) repeat sequences that contain palindromic sequences that can fold over to form hairpin structures that function as primers during initiation of DNA replication. In addition to their role in DNA replication, the ITR sequences have been demonstrated to be necessary for viral integration, rescue from the host genome and encapsidation of viral nucleic acid into mature virions (Muzyczka, N., Curr. Top. Micro. Immunol. 158:97-129, 1992).
The capsids have icosahedral symmetry and are about 20-24 nm in diameter. They are composed of three proteins (VP1, VP2, and VP3, which are approximately 87, 73 and 61 Kd, respectively) (Muzyczka, N., Curr. Top. Micro. Immunol. 158:97-129, 1992). VP3 represents 90% of the total virion protein; VP2 and VP1 account for approximately 5% each. All capsid proteins are N-acetylated.
RECOMBINANT PRODUCTION OF VIRAL CAPSIDS
Recombinant DNA technology has been used to isolate the genes for structural proteins of many viruses. For example, vaccinia virus has been used to carry the structural genes for human papilloma virus 1 (Hagensee, M. E., et al., J. Virol. 67:315-322, 1993); simian immunodeficiency virus (Gonzalez, S. A., et al., Virology 194:548-556, 1993); and Aleutian mink disease parvovirus (Clemens, D. L., et al., J. Virol. 66:3077-3085, 1992); capsid formation has been detected in all systems. Baculovirus vectors have been used for the expression of the structural proteins of human papilloma virus (Kirnbauer, R., et al., J. Virol. 67:6929-6936, 1993) and B19 parvovirus (Kajigaya, S., et al., Proc. Natl. Acad. Sci. 88:4646-4650, 1991); these proteins have assembled into capsids within the infected cells. Baculovirus-mediated expression of the capsid proteins of adeno-associated virus-2 (Ruffing, M., et al., J. Virol. 66:6922-6930, 1992) resulted in the formation of capsids with an altered stoichiometry from wild-type capsids and which failed to localize into the nuclear clusters observed in a wild-type infection.
These efforts have been expended to study virus life-cycles and do not use of any of these systems to encapsidate foreign genomes or other materials for delivery in vivo.
SUMMARY OF THE INVENTION
The invention relates to the production of AAV capsids which may be used to transfer native or heterologous molecules into appropriate host cells. The capsid proteins can be expressed from a recombinant virus, expression vector, or from a cell line that has stably integrated the AAV capsid genes or coding sequences. The invention further provides for the production of AAV capsids in vitro from the AAV capsid proteins and the construction of packaged capsids in vitro. The invention further provides for the production of AAV capsids that have been genetically engineered to express heterologous epitopes of clinically important antigens to elicit an immune response.
Molecules which may be associated with or encapsidated into capsids include DNA, RNA, proteins, peptides, small organic molecules, or combinations of the same. The AAV capsids can accommodate nucleic acids which are quite large e.g., 5000 bp, and therefore, may be advantageously used for the transfer and delivery of large genes and genomic sequences. Because the AAV inverted terminal repeats (ITRs) are responsible for the ability of the AAV genome to integrate into the host cell genome (Samulski, R. J., et al., EMBO J. 10:3941-3950, 1991), these sequences may be used with the heterologous DNA in order to provide for integration of the heterologous DNA into the host cell genome and may further facilitate packaging into an AAV capsid.
The invention is demonstrated by way of examples in which the AAV capsid is produced from a recombinant adenovirus engineered to carry the capsid genes. This system may be particularly advantageous in AAV gene delivery systems because adenovirus serves as a natural helper for AAV infection. Upon infection of the host cell, expression of the capsid
Ferrari Forrest K.
Samulski Richard Jude
Martinell James
Pennie & Edmonds LLP
University of Pittsburgh
LandOfFree
AAV capsid vehicles for molecular transfer does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with AAV capsid vehicles for molecular transfer, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and AAV capsid vehicles for molecular transfer will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2464807