Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
1999-01-11
2002-06-25
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S235100, C435S320100, C435S005000, C424S093200
Reexamination Certificate
active
06410300
ABSTRACT:
FIELD OF THE INVENTION
This invention relates t o methods and formulations for mediating virus attachment and infection, and more particularly relates to methods and formulations for mediating adeno-associated virus attachment and infection.
BACKGROUND OF THE INVENTION
Adeno-associated virus (AAV) is a human parvovirus that infects a broad range of cell types including human, non-human primate, canine, murine, and avian. A member of the Parvoviridae family, AAV is a small non-enveloped single-stranded DNA virus of 20-25 nm which has an unique requirement for a helper virus (e.g., adenovirus or herpes simplex virus) to complete its lytic cycle (R. W. Atchison et al., (1965)
Science
149:754; M. D. Hoggan et al, ((1966)
Proc. Natl. Acad. Sci. USA
55:1457; J. L. Melnick et al., (1965)
J. Bacteriol
. 90:271). In the absence of helper virus, AAV still infects the target cell, but integrates into the host genome and establishes latency. Unique among eukaryotic DNA viruses, the AAV genome can integrate site specifically into human chromosome 19 (R. M. Kotin et al., (1990)
Proc. Natl. Acad. Sci. USA
87:2211; R. J. Samulski et al., (1991)
EMBO J
. 10:3941; R. J. Samulski, (1993)
Curr. Opin. Genet. Dev
. 3:74; C. Giraud et al., (1994)
Proc. Natl. Acad. Sci. USA
91:10039; C. Giraud et al., (1995)
J. ViroL
69:6917). This property has drawn considerable attention to the potential use of AAV as a gene therapy vector, although little is known about the initial events of AAV infection (R. J. Samulski, (1995) Adeno-associated virus-based vectors for human gene therapy, p. 232-271. In K. M. Hui (ed.), Gene therapy: from laboratory to the clinic. World Scientific Publishing Co., Singapore, Singapore; C. McKeon et al., (1996)
Hum. Gene Ther
. 7:1615; D. M. McCarty et al., (1997) Adeno-associated viral vectors, p. 62-78. In M. Strauss and J. Barranger (ed.), Concepts in gene therapy. Walter de Gruyter, Bellin. N.Y.; R. J. Samulski, (1997) Development of adeno-associated virus as a vector for in vivo gene therapy, p. 197-203. In L. M. Houdebine (ed.), Transgenic animals: generation and use. Harwood Academic Publishers, Chur, Switzerland). In particular, the recombinant AAV or rAAV vector system is well characterized and is the subject of increasing development as a vector for gene delivery (see, C. McKeon et aL (1996)
Hum. Gen. Ther
. 7:1615). In general, AAV vectors are generated by deleting rep and cap genes and replacing them with genes intended for delivery into the cell. Additionally, producer cells that contain rep and cap may be used to package the gene therapy vectors into the AAV capsid particle (B. J. Carter, (1996)
Nature Biotechnology
14:1725).
Despite this growing interest in AAV, the events that govern the initial AAV infection remain poorly understood. The primary event of any viral infection is attachment of virus to the host cell. A wide variety of cell surface molecules are now known to serve as viral attachment receptors. However, the mechanism by which AAV attaches to its host cell has heretofore not been delineated. AAV has a very broad host range and infects a wide variety of cell types, suggesting that the virus uses a ubiquitous receptor to mediate infection. Identification of the initial virus-host cell interactions necessary for efficient AAV infection is not only important for the general understanding of parvovirus infection, but also for the effective use of AAV as a gene therapy vector.
Although the initial events in the life cycle of AAV are not well understood, previous studies suggest that AAV infects cells through interaction with a specific host cellular receptor (H. Mizukami et al., (1996)
Virology
217:124; S. Ponnazhagan et al, (1996)
J. Gen. Virol
. 77:1111). AAV appears to exhibit saturation binding to HeLa cells. In addition, cellular attachment of AAV is sensitive to trypsin treatment, suggesting a protein component is responsible for binding. Id.
The lack of knowledge concerning the receptor of AAV has introduced significant obstacles to the development of reliable techniques for both isolating and using AAV as a means for gene therapy. For example, purification of AAV is generally conducted using techniques that ultimately involve the use of a CsCl gradient. There are certain disadvantages in using these techniques, primarily because CsCl is toxic and thus requires special handling. It would be highly desirable to develop a milder and less dangerous means of isolating AAV viral particles.
An additional obstacle to the use of AAV as a reliable gene therapy vector has been the difficulty in infecting certain types of cells with the vector. Experiments in cultured cells have shown that AAV vectors are efficient for delivery of genes to both dividing and non-dividing cells. However, these experiments have also shown that the efficiency and both expression and metabolic activation may vary with the cell type and the physiological state of the cell (C. McKeon et al., (1996)
Hum. Gen. Ther
. 7:1615). In particular, progenitor or stem cells (e.g., bone marrow CD34
+
cells) have been found to be difficult to infect with the AAV vector. Additionally, in some cell types, persistence and expression of a heterologous gene carried by the vector are not well maintained. Finally, even when it is known that certain cell types are generally permissive to infection by AAV, is appears that there is diversity among individual cell donors as to whether or not any particular donor's cells will permit infection by the AAV vector. It would be highly desirable to have means for the effective infection of stem cells and rare cell types, as well as the means for introducing the AAV vector into cells that may not naturally express the AAV receptor, or may not naturally produce the molecular substituents necessary for the attachment and internalization of the virus.
Accordingly, there is a need in the art for improved methods and reagents for purifying AAV and rAAV vectors. In addition, there is a need in the art for methods of modifying the wild-type tropism of AAV vectors for use in gene therapy and for screening cells for permissiveness to transduction by AAV vectors.
SUMMARY OF THE INVENTION
The methods, AAV vectors, and formulations of the present invention are based on the surprising discovery that has identified cell surface heparin and heparan sulfate (HS) proteoglycan as the primary cellular receptors for AAV. It has also been discovered that AAV interacts specifically with cell surface heparin and heparan sulfate glycosaminoglycans (GAG), and not other glycosaminoglycans. Further, it has now been determined that the presence of HS GAG on the cell surface directly correlates with the efficiency by which AAV can infect cells.
Moreover, a role has been established for &agr;
v
&bgr;
5
integrin in AAV infection. AAV virions physically interact with the &bgr;
5
subunit of &agr;
v
&bgr;
5
integrin. Using genetically defined cell lines that either lack or express &agr;
v
&bgr;
5
, it has been demonstrated that cell surface expression of this integrin promotes AAV infection. The present investigations suggest that &agr;
v
&bgr;
5
integrin acts to facilitate the internalization of AAV bound to cell surface heparin and HS proteoglycans into the cell. This is the first report of the involvement of an integrin in a parvovirus infection.
These discoveries have led to the development of methods and formulations that mediate the infection of a broad range of cell types with AAV, including cells that are typically non-permissive for infection by AAV. Additionally, these discoveries have led to the development of methods of purifying AAV using receptor-like molecules that bind to AAV, and methods of screening cell samples for their permissiveness to infection with AAV. Furthermore, these discoveries have elucidated new strategies for modifying the natural tropism of AAV, in particular, for use in gene therapy.
Accordingly, a first aspect of the present invention is a method of facilitating attachment of AAV to a cell, and infection of a cell by AAV, by conta
Samulski Richard Jude
Summerford Candace
Myers Bigel Sibley & Sajovec P.A.
Priebe Scott D.
The University of North Carolina at Chapel Hill
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