Dept. of Ophthalmology and Visual Sciences

University of Wisconsin

Madison, WI

MURFEE MACULAR DEGENERATION PROJECT

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 cell therapy, 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.

Plans for 2024        

Purpose of Project: 
The goals of this project are to understand the innate immune responses to viral gene delivery vectors (gdv), and reduce the impact of host cell restriction factors on transduction efficiency, to improve gene therapy for ocular diseases.

Dr. Brandt will continue his research on the early inflammatory response of human retinal organoids to AAV gene delivery vectors.  We will also begin a project in human retinal cell lines to examine the effect of a human cytomegalovirus protein, which inhibits cell death and immune signaling, on innate immune sensor proteins, gdv transduction efficiency, and cell survival.

Specific Aims:

Aim 1. To continue our studies on the early inflammatory response of human retinal organoids to AAV in order to understand how this vector induces uveitis.

Aim 2. To examine the effect of the human cytomegalovirus viral mitochondria-localized inhibitor of apoptosis (vMIA) protein on innate immune sensor proteins, gdv transduction efficiency, and cell survival in human retinal cell lines.

Progress in 2023

Dr. Brandt completed and published his lab’s studies on RNA sensor protein expression in neural retina tissue and identified a mitochondrial sensor protein that restricts viral gdv transduction in human Mueller cells (Sauter et al., 2023). They also completed an analysis of the effect of  viral gdv transduction on RNA and DNA sensor gene expression in human retinal cell lines. The lab explored whether small molecule transcription factor activators and inhibitors altered transduction efficiency of viral gdvs in retinal cells. Initial experiments investigating the inflammatory response of human retinal organoids to AAV detected changes in gene expression and secretion of proteins that may contribute to inflammation during ocular gene therapy.

Progress in 2022

Dr. Brandt examined the effect of activation of a common transcription factor on gdv efficiency in two human retinal cell lines. Hi lab determined the expression and cellular distribution of host cell restriction factors, that recognize viral RNA and DNA, in human retinal cell lines and non-human primate (NHP) retina tissue.  They also demonstrated that knockdown of a mitochondrial protein linked to host cell restriction factor signaling could significantly increase gdv efficiency in a human Muller cell line.

Specific Aims: Aim 1. To determine if NFκB activation can increase viral gene delivery vector efficiency in human retinal cells. Aim 2. To examine the role of host cell cytoplasmic RNA and DNA sensors in viral gene delivery in human retinal cells. Aim 3. To explore the function of TRIM5α in a human Muller cell line.

Progress in 2021

Progress in 2021: Dr. Brandt’s laboratory completed their studies to determine whether exposure to viral gene delivery vectors (gdvs) altered the expression of key inflammasome genes in macaque retina tissue and two human retinal cell lines. They also performed assays to screen for secreted cytokines in supernatants of gdv challenged retina tissue and retinal cell lines. The lab team completed and published our results on the effect of proteasome inhibition on viral vector transduction efficiency in a human Muller cell line. They explored the role of TAK1 kinase activation in restriction of viral vectors in Muller cells. Finally, they also identified a link between activation of a key transcription factor and knockdown of two host cell restriction factors in human Muller cells.

Progress in 2020

Dr. Brandt completed his study of inflammasome gene expression in gene delivery vector (gdv) challenged NHP retina tissue and human Muller cells.  His research team examined the localization of two host restriction factors in NHP retina tissue and human Muller cells and also determined that a decrease in these restriction factors increased the transduction efficiency of a viral gdv in human Muller cells.  Dr. Brandt documented a change in the mobility of a kinase involved in innate immune signaling after gdv challenge of Muller cells.

Progress in 2019

Dr. Brandt’s lab determined that NHP retina tissue expresses many components of the inflammasome pathway in a variety of retinal cell types.  Expression of several inflammasome pathway components was increased following viral vector  challenge of human Muller cells.  They also determined that a decrease in host restriction factors, which resulted in an elevated level of reverse transcription, increased the transduction efficiency of a viral gene delivery vector in human Muller cells.

Progress in 2018

In 2018 Dr. Brandt’s research team determined that non-human primate retina tissue expresses many components of the inflammasome pathway in a variety retinal cell types. In addition, they determined that a decrease in host restriction factors increased the transduction efficiency of a viral gene delivery vector in a human Muller cell line.

The project’s continuing specific aims are:
1. To identify the expression pattern of inflammasome components in the nonhuman primate retina and to determine whether expression of these genes is altered by gene delivery vector challenge. 2. To determine whether non-human primate retina tissue produces host restriction factors and to study the effect of host restriction factors on gene delivery vector replication in a retinal cell line.

Progress in 2017

The purpose of 2017 research was to determine the mechanism of inflammation triggered by viral gene delivery vector injection in the non-human primate eye.  The expression of innate immune receptors were examined, including Toll-like receptors and non-self nucleic acid receptors, as well as components of the inflammasome in non-human primate retina.

Progress in 2016

Specific aims accomplished in 2016 included: 1) Compared gene expression profiles between Mauritius macaque retina tissue before and after viral vector challenge; Dr. Brandt continued work focused 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 2015

Research conducted in 2015: 1) Compared gene expression profiles between Mauritius macaque retina tissue before and after viral vector challenge; in future experiments, Dr. Brandt planned to focus on cynomolgus and rhesus macaques; 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

Dr. Brandt’s research: 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 micro-array to examine the expression of innate immune molecules and inflammatory cytokines.

Progress in 2013

Dr. Brandt’s experiments conducted in 2013 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 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.

Photos by Andy Manis

HELMERICH CHAIR from 7/1/10 – 6/30/12

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.