AAV capsid vehicles for molecular transfer

Abstract

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.

Description

1. INTRODUCTION[0001] 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.2. BACKGROUND OF THE INVENTION[0002] 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.2.1. PHYSICAL TRANSFER METHODS[0003] 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 t...

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Benefits of technology

0014] 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 A

Problems solved by technology

These techniques are utilized in the research setting but require in vitro handling of the recipient cells and thus have limited clinical potential.

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.

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.

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