Preserving the Precious Gift of Sight

Curtis Brandt, PhD

MURFEE MACULAR DEGENERATION PROJECT

Dept. of Ophthalmology and Visual Sciences

University of Wisconsin

Madison, WI

 

Dr. Brandt’s Research Project

“Gene Therapy for Retinal Degenerative Diseases”
 

Current Research Interests

Retinal degenerative diseases such as retinitis pigmentosa (RP) and macular degeneration (MD) are significant causes of blindness in the United States. To date there are few therapies for these diseases, but a number of approaches are under investigation including retinal transplantation, stem cells, and gene therapy. Several different viruses have been utilized as gene delivery vectors including herpes simplex virus (HSV), adenovirus (AdV), adeno-associated virus (AAV), and lentiviruses. Many factors must be considered when designing a vector for ocular gene delivery including selection of viral vector, delivery route, cellular target, and choice of promoter. Dr. Brandt’s work in rodents showed that gene delivery with HSV vectors did not induce inflammation of the eye. In contrast, he found that adenovirus and lentiviral vectors induced a transient inflammatory response in primate eyes. The ultimate goal of this project is to develop a strategy for preventing viral vector induced inflammation in the primate eye in order to improve gene therapy for human ocular diseases. Understanding the mechanisms triggering these immune responses will lead to strategies to reduce viral vector induced transient uveitis.

Progress in 2016

1) Compared gene expression profiles between Mauritius macaque retina tissue before and after viral vector challenge; Dr. Brandt will focus on cynomolgus and rhesus macaques in future experiments; 2) Performed RNA in situ hybridization on macaque neural retina tissue exposed to the HSV-1 vector hrR3; confirmed that IL-6 was expressed in both photoreceptor and Mueller cells; 3) Evaluated the inflammatory response of retinal pigment epithelial (RPE) cells following exposure to viral gene delivery vectors; Dr. Brandt had expected that more cytokines would be secreted following viral vector challenge of RPE cells.

Plans for 2017

The goal of this ongoing project research is to continue to study innate immune responses to viral vectors and identify the mechanisms involved in triggering transient uveitis in the retina. Specific Aims: 1) To examine the interaction between HSV-1 glycoprotein gB and TLR2 in Muller cells to determine its role in triggering NFKB activation and cytokine synthesis; 2) To determine the expression profile of Toll-Like Receptor proteins 1 through 10 in NHP neural retina tissue; 3) To determine the expression pattern of non-self nucleic acid receptor, including retinoic acid-inducible gene (RIG)-I-like receptors and cytosolic DNA sensors (CDSs), and inflammasome components in the NHP retina; 4) To determine whether NHP retina tissue produce host restriction factors that may reduce the transduction efficiency of viral gene delivery vectors.

Progress in 2015

1) Compared gene expression profiles between Mauritius macaque retina tissue before and after viral vector challenge; Dr. Brandt will focus on cynomolgus and rhesus macaques in future experiments; 2) Performed RNA in situ hybridization on macaque neural retina tissue exposed to the HSV-1 vector hrR3; confirmed that IL-6 was expressed in both photoreceptor and Mueller cells; 3) Evaluated the inflammatory response of retinal pigment epithelial (RPE) cells following exposure to viral gene delivery vectors; Dr. Brandt had expected that more cytokines would be secreted following viral vector challenge of RPE cells.

Progress in 2014

1) Determined, by Western blotting, the expression levels of innate immune response molecules in monkey retina tissue; 2) Examined the expression and distribution of inflammasome components in macaque retina tissue; 3) Analyzed RNA isolated from macaque retina tissue before and after viral vector challenge by quantitative PCR microarray to examine the expression of innate immune molecules and inflammatory cytokines.

Progress in 2013

Dr. Brandt’s recent experiments implied that HSV-1 may be utilizing the Toll-Like Receptor 9 (TLR9) signaling pathway to activate NFkB during its replication cycle. More recent experiments with inhibitory TLR9 oligonucleotides indicate that the oligos decreased viral replication in both TLR9 positive and TLR9 negative cells. Experiments now indicate that these TLR9 inhibitory oligos may be acting during early stages of the viral life cycle, such as attachment and entry, through an anti-viral mechanism.

Progress in 2012

Dr. Brandt’s lab has demonstrated that upon challenge with HSV-1, or the HSV-1 gene delivery vector hrR3, IL-6 is induced in retinal tissue. During previous studies, they determined that the transcription factor NFKB was activated in nuclei within the inner nuclear layer of retina tissue upon virus challenge. These results are intriguing as they are not seeing IL-6 expression in the same retinal cells in which NFKB is activated. The means by which these cells may communicate to produce IL-6 in neighboring cell is unknown. Further studies into the distribution of innate immune markers in the retina may help explain this phenomenon.

 

HELMERICH CHAIR from 7/1/10 – 6/30/12

Dr. Brandt’s laboratory is focusing on using viral based vectors for ocular gene delivery. They and others have shown that vectors based on several different viruses can deliver genes to various cells in the retina. Other work in his laboratory and in collaboration with others has shown that introduction of many of these viral vectors into the primate eye triggers a transient inflammatory response. Interestingly, inflammatory responses are not triggered in rodent eyes.

For ocular gene therapy to move forward in people, it is necessary to identify the cause of the inflammatory response so strategies to block the effect can be developed. Dr. Brandt’s lab is studying several pre-inflammatory signaling molecules that could be the signal that initiates the process.

Because many viruses are human pathogens, our host defense systems can be activated even when replication defective viruses are used for gene delivery. Our bodies have “innate” recognition systems that can see these viral vectors and when they recognize the presence of a vector they trigger a defensive response.  This response has a number of negative consequences that can affect therapeutic use of the vector.  These include the activation of an immediate inflammatory response that can cause pathology or negatively affect the efficiency of the gene delivery.

Dr. Brandt’s laboratory has recently found that the activation of some of these defense systems are actually required for efficient replication of viruses in the retina, raising the possibility that these defense systems might affect the efficacy of viral gene delivery.  They are currently studying how these systems are activated by viral gene delivery vectors and whether this has an effect on gene delivery.  In particular, one particular receptor system seems to be required for expression of the very early herpes simplex virus proteins that are essential for replication.

Photos by Andy Manis
Photos by Andy Manis

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Mission of RRF

The mission of the Retina Research Foundation is to reduce retinal blindness worldwide by funding programs in research and education. As a public charity, RRF raises funds from the private sector and the investment of its endowment funds.

RRF’s 48th Anniversary

October 1, 2017