Next generation gene therapy

PHILADELPHIA – Inspired by earlier successes using gene therapy to correct an inherited type of blindness, investigators from the Perelman School of Medicine at the University of Pennsylvania, are poised to extend their approach to other types of blinding disorders.

In a previous human trial conducted at the Children's Hospital of Philadelphia and Penn, researchers packaged a normal version of a gene missing in Leber's congenital amaurosis (LCA) inside a genetically engineered vector, called an adeno-associated virus (AAV). The vector delivered the gene to cells in the retina, where the gene produces an enzyme that restores light receptors.

"The results from three Phase I clinical trials for LCA showed the potential for gene therapy based on adeno-associated viruses delivering corrective genes to the retina," notes co-senior author Jean Bennett, MD, PhD, the F.M. Kirby professor of Ophthalmology. "To broaden treating inherited eye diseases, we will need a larger vector toolkit, and what we have seen of AAV8 gives us hope for creating gene therapies for diseases that attack the retina's photoreceptors. This preclinical study provides the guidance we need to formulate dose level and type of vector to deliver corrective genes to treat blindness caused by the loss of photoreceptors."

In the present study, published in Science Translational Medicine this week, the Penn team compared the safety and efficiency of delivery in an animal model of two different types of AAVs - AAV2, which was used in the human trials for LCA, and AAV8, a second-generation AAV technology initially discovered in the lab of co-senior author James M. Wilson, MD, PhD, professor of Pathology and Laboratory Medicine.

The researchers used both vectors to deliver a green fluorescent protein (GFP) transgene to the retinal pigment epithelial (RPE) cells and photoreceptor cells of nonhuman primates. Photoreceptor cells are the problem area for other retinal diseases such as retinitis pigmentosa (RP) and others, for which there is no treatment. Photoreceptors are specialized nerve cells that convert light into a biological electrical signal and are designated as rods and cones.

"We showed that we can use AAV8 to deliver genes to the photoreceptor of the primate eye at lower doses, both safely and efficiently," says first author Luk H. Vandenberghe, PhD, Senior Investigator, Gene Therapy Program, and currently at Penn's F.M. Kirby Center for Molecular Ophthalmology.

Both AAV2 and AAV8 delivered the gene safely and efficiently to the monkey retinas, but AAV8 was markedly better at targeting photoreceptor cells.

The STM study describes the dose relationship between AAV2 and AAV8 vectors and their target cells and the immune response in the nonhuman primate retina. From this the researchers found dosage thresholds to safely and efficiently target cells in the outer retina such as RPE cells and rod and cone photoreceptors. While AAV2 and AAV8 efficiently delivered the gene to RPE cells at moderate to low doses, expression of the GFP gene in rod and cone photoreceptors was reached only at higher dosages. Substantial delivery to rods was obtained with moderate doses of AAV8, doses similar to those currently used in experimental clinical protocols.

The vectors at intermediate doses did not cause problematic immune responses and post-surgical injection complications. The delivered gene also stayed in the retina at very high levels throughout the study duration of four months. Additionally, the GFP gene was preferentially transduced to one type of retinal ganglion cells, which surprised the researchers. In general retinal ganglion cells transmit visual information from the retina to several regions of the brain. This knowledge could be used in the future to further delineate the neuronal connections between the retina and the brain.

In earlier animal studies, AAV8 also delivered genes safely and efficiently to the mouse retina. However, the mouse retina differs significantly from the primate retina, most notably in structure, which affects the surgical approach to delivering corrective genes to different parts of the eye. (For example, mice do not have a macula – the eye structure used for visual discrimination, which is affected in macular degeneration – and primates do.) The present study in nonhuman primates is the next step to better translate treatment strategies for people.

"To address patients with other retinal diseases, we need a renaissance of technology--new and better vectors to safely and effectively deliver corrective genes to a range of diseases," says Wilson. "My lab has recently isolated new families of simian-based adenoviruses and adeno-associated viruses. Recombinant versions of these viruses are turning out to be useful as improved gene transfer vehicles to a variety of targets."

The other co-authors, all from Penn, are Peter Bell, Albert M. Maguire, Cassia Cearley, Ru Xiao, Roberto Calcedo Lili Wang, Michael J. Castle, Alexandra C. Maguire, Rebecca Grant, and John H. Wolfe. Maguire, Bennett, and Wolfe also have affiliations with The Children's Hospital of Philadelphia.

The study was supported by grants from GlaxoSmithKline Pharmaceuticals Inc.; Foundation Fighting Blindness, Research to Prevent Blindness; the Paul and Evanina Mackall Foundation Trust; the F. M. Kirby Foundation; the National Institute of Neurological Disorders and Stroke; the National Center for Research Resources; and the Institute for Translational Medicine and Therapeutics at the University of Pennsylvania.

Editors Note

Wilson is a consultant to ReGenX Holdings and is a founder of, holds equity in, and receives a grant from ReGenX Biosciences. Wilson and Vandenberghe are inventors on patents related to AAV8 technology that are owned by Penn and licensed to GlaxoSmithKline and for some rights sublicensed to a number of biopharmaceutical companies including ReGenX Biosciences. Wilson is an inventor on a patent licensed to various biopharmaceutical companies (adeno-associated virus serotype 8 sequences, vectors containing same, and uses thereof; USPTO 20080075740, WO/2003/052051). Maguire and Bennett are co-inventors on a pending patent for a method to treat or slow the development of blindness (WO/2002/082904, PCT/US2002/011314); both waived any financial interest in this technology in 2002. Bennett served on a scientific advisory board for Sanofi-Aventis and as a consultant for GlaxoSmithKline in 2010. The other authors declare no competing interests.

Penn Medicine is one of the world's leading academic medical centers, dedicated to the related missions of medical education, biomedical research, and excellence in patient care. Penn Medicine consists of the Raymond and Ruth Perelman School of Medicine at the University of Pennsylvania (founded in 1765 as the nation's first medical school) and the University of Pennsylvania Health System, which together form a $4 billion enterprise.

Penn's Perelman School of Medicine is currently ranked #2 in U.S. News & World Report's survey of research-oriented medical schools and among the top 10 schools for primary care. The School is consistently among the nation's top recipients of funding from the National Institutes of Health, with $507.6 million awarded in the 2010 fiscal year. The University of Pennsylvania Health System's patient care facilities include: The Hospital of the University of Pennsylvania -- recognized as one of the nation's top 10 hospitals by U.S. News & World Report; Penn Presbyterian Medical Center; and Pennsylvania Hospital – the nation's first hospital, founded in 1751. Penn Medicine also includes additional patient care facilities and services throughout the Philadelphia region.

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