ReGenX’s technology platform derives from discoveries in the laboratory of Professor James M. Wilson, M.D., Ph.D. at the University of Pennsylvania that led to identification of at least 30 new serotypes of adeno-associated virus (AAV). Several of these novel serotypes, including AAV7, AAV8, and AAV9 have emerged as vectors for gene delivery which are dramatically more efficient than previous serotypes. The ReGenX AAV Vector Technology enables the efficient, rapid, safe and specific delivery of genetic material into cells. Once delivered into a cell, the genetic material can use the existing cellular machinery to produce specific proteins encoded by the genetic material.

This process may be used to treat disease by initiating normal protein production or gene regulation in cells that may have defects in their endogenous function. Delivery of genetic material may also enable cells to produce more of a certain protein or different proteins than they would normally produce, thereby treating a disease state. Finally, the ReGenX AAV Vector Technology can also be used to deliver specific sequences of genetic material that, once delivered and expressed, can turn off or interfere with the production of disease specific genes and their active protein products.

ReGenX AAV Vector Technology Applications

Category	Feature	Sample Molecular Therapy/Disease
Gene Therapy	Replace missing or defective protein	RPE65/Leber Congenital Amaurosis
	Increase levels of necessary protein	Factor IX/Hemophilia B
	Add therapeutic protein continuously	anti-TNFR mAb/Rheumatoid Arthritis
RNAi	Decrease levels of problem protein	VEGF/Cancer
Stem Cell Therapy	Promulgate change in cell phenotype	Many ex-vivo or in-vivo applications including diabetes and heart failure

Delivery of Genetic Material to Cells

The primary issue with delivering genetic material to cells is how to get the large, highly charged DNA or RNA to enter cells and reach the nucleus efficiently. This can be accomplished with purified DNA by using various transfection techniques, such as calcium phosphate precipitation, electroporation, and liposomes. While these transfection techniques can be efficient in vitro, they are typically inefficient in vivo. An alternative method is to use engineered viruses, as viruses have evolved to efficiently deliver their genetic payload to cells. A comparison of different in vivo gene delivery techniques is shown in the table below.

In Vivo Gene Delivery Technologies

Vector	Cloning Capacity*	Integration**	Non-Dividing	Inflammatory Response	Expression Levels	Stable Expression
DNA	–	Low	Yes	Low	Low	No
Retrovirus	~4 kb	High	No	Low	Low	Yes
Lentivirus	~8 kb	High	Yes	Low	Low–Medium	Yes
Adenovirus	>8 kb	Low	Yes	High	High	No
AAV	~4.5 kb	Low	Yes	Low	Low	Yes
ReGenX AAV Vector Technology	~4.5 kb	Low	Yes	Low	High	Yes

 * Capacity refers to the maximum amount of foreign DNA that can be inserted
** For retrovirus and lentivirus, integration could be associated with activation of oncogenes
“Non-dividing cells” refers to the capacity to deliver genetic material to cells that do not divide.

Adeno-Associated Virus (AAV)

Electron micrograph of purified AAV
(courtesy P. Bell, University of Pennsylvania)

AAV is a parvovirus, 18–26 nm in diameter, consisting of only protein and DNA. Its protein coat contains three capsid proteins enclosing its single-stranded DNA genome. AAVs were initially isolated in cultures associated with adenovirus, and at the time only 6 human serotypes were identified (AAV1-AAV6). AAVs are naturally replication-defective, requiring helper genes from another virus such as adenovirus or herpes simplex virus to replicate. Interestingly, despite the fact that most humans have been exposed to AAV, AAVs have not been associated with any disease.

The AAV genome consists of two inverted terminal repeats (ITRs) flanking the two AAV genes, rep (encoding replication proteins) and cap (encoding capsid proteins). Recombinant AAV (rAAV) deletes both rep and cap, and instead inserts an expression cassette between the two ITRs. Before the discovery of additional, novel, serotypes, AAV2 was the serotype most commonly used to make recombinant AAV. The AAVs used in ReGenX AAV Vector Technology are hybrids, using the AAV2 ITRs (and AAV2 rep genes for production) with the capsid derived from the desired serotype. Such a hybrid is designated AAV2/8 to indicate that the capsid proteins are from AAV8.

Abbreviations:

ssDNA genome: single-stranded DNA genome of AAV
rep: replication gene
cap: capsid gene
ITR: inverted terminal repeats. These are the ends of the linear, single-stranded AAV genome.
expr. cassette: expression cassette. To express cDNAs, the cassette would contain a promoter, an intron (optional), the cDNA, and a polyadenylation site.

These recombinant, replication defective AAVs are able to enter cells and deliver their DNA genome to the nucleus, but are unable to replicate. AAVs (both wild-type and recombinant) bind initially to cell-surface receptors, some of which have been identified. AAV2 binds to heparan sulfate proteoglycans¹, and uses a co-receptor ^2,3, while AAV4 and AAV5 bind to sialic-acid containing glycoproteins⁴. When the AAV genome reaches the nucleus, the single-stranded DNA must be converted to double-stranded DNA before gene expression will occur. This process can take some time in vivo, so that transgene expression from rAAV may not reach a maximum until 1–6 weeks after injection. Most of the rAAV genomes do not integrate, but are maintained episomally⁵. In non-dividing or slowly dividing cells in vivo, transgene expression may persist for years.

References