Gene and Cell Therapy Institute (GCTI) Awards

Evaluation of human miniPCDH15s in Human Inner Ear Organoids to treat USH1F

Usher syndrome type 1F (USH1F) causes deafness and blindness due to mutations in the protocadherin-15 gene (PCDH15). A simple gene addition therapy for USH1F remains challenging because the PCDH15 coding sequence is too large for adeno-associated virus (AAV) vectors. To overcome this limitation, the team developed “mini”-PCDH15 versions that fit into a single AAV capsid and can restore hearing and balance in mouse models of USH1F. Dr. Indzhykulian will test similarly designed human mini-PCDH15 versions to evaluate their function in human inner-ear organoids. This study will provide a proof-of-concept, preclinical evaluation of human mini-PCDH15s, an essential step for clinical translation.

AAV capsids which efficiently cross the blood brain barrier in primates

Two distinct, yet intertwined major problems are faced by adeno-associated virus (AAV) vectors for global treatment of diseases that affect the brain. First, the standard of care serotype for gene therapy to the CNS, AAV9, crosses the blood-brain barrier (BBB) at relatively low efficiency, so high doses are required for therapeutic benefit. AAV vectors (like other biologics) are costly and difficult to make to treat larger cohorts of patients. The second problem, which is linked to high doses of vector, is activation of the innate and adaptive immune system, which in some cases has led to clinical holds owing to sepsis-like symptoms and even organ failure and death. AAV peptide display libraries to discover novel capsids have been used successfully to achieve efficient transduction of murine CNS after systemic delivery. These proof-of-concept studies show promise that efficient AAV capsids may solve both issues above. The main issue that remains to be solved is obtaining “translationally relevant” AAV capsids that work in humans, as the capsids that have been selected in mice generally do not work in non-human primates (NHPs) and likely won’t work in humans to cross the BBB. The team’s solution is to perform a selection which results in identification of capsids with a high likelihood of crossing the BBB in humans and transducing target neurons.

Rapid identification of nanobody chimeric antigen receptors using nanomice

The team has developed a high-throughput method for generating nanobody-based chimeric antigen receptors (CAR) targeting any antigen. Dr. Manguso’s platform uses a novel strain of mice that produce single-domain (VHH) antibodies. Through immunization of these mice with dendritic cells expressing an antigen of interest, we can induce robust antigen-specific B-cell expansion. From there, the team directly isolates and clones VHH libraries from the immunized mice into CAR vectors and selects for highly functional and specific. This method has been applied to generate novel nanobody-CARs for mesothelin and is currently being used for the development of CARs targeting several additional antigens.

A "One-and-Done" CRISPR therapeutic for multiple repeat expansion diseases

The team is developing a novel CRISPR base editing therapeutic that specifically targets somatic repeat instability, which is a hallmark of disease in multiple repeat expansion disorders, including Huntington’s Disease, Friedreich Ataxia, Myotonic Dystrophy, X-Linked Dystonia Parkinsonism and multiple Spinocerebellar Ataxias. More specifically, we are targeting the DNA repair pathway to prevent pathogenic somatic expansion of disease-causing trinucleotide repeats. This therapeutic reagent would be delivered to disease vulnerable tissues as a single dose to effectively prevent disease onset or halt symptoms' progression.

Nociceptor-specific gene therapy for chronic pain

The team is creating highly specific adeno-associated viruses (AAV) for long-term pain management. The therapy involves using a chemogenetic actuator, activated by an FDA-approved drug, to silence target nociceptors in patients who experience temporary relief from nerve blocks. To avoid anesthesia from off-target neuronal silencing, this approach will leverage gene regulatory elements the lab has identified to promote AAV expression specifically in nociceptors. This one-time therapy aims to provide patient-controlled local analgesia based on existing outpatient interventional nerve block procedures. Moreover, the general approach offers a platform for further development of cell-type and cell state-specific AAVs that may be applicable to other conditions.

Focused ultrasound for delivery of AAV vectors across the BBB

The goal of this project to validate the use of focused ultrasound (FUS) blood-brain barrier (BBB) opening combined with systemic administration of a novel AAV capsid as a platform technology for delivering gene therapies to the brain. FUS can be applied to achieve non-invasive and temporary permeabilization of the BBB in targeted brain regions, allowing normally non-penetrant therapies to reach the brain from systemic circulation. Preliminary data in mice indicates that particularly potent neuronal transfection can be achieved when FUS- BBB opening is combined with a novel AAV capsid, AAV.CPP, engineered for improved penetrance across the BBB. The team will characterize delivery efficacy relative to the current state of the art and demonstrate proof-of-concept gene knock-down/replacement in two clinically relevant diseases. It is anticipated that data from these studies will showcase FUS-BBB opening + AAV.CPP as a leading platform technology for delivering gene therapies to the brain, allowing for several paths to further develop and translate this exciting technology.

Developing precision gene therapy treatment for severe pain

Current chronic pain management approaches offer limited benefits and cause substantial systemic side effects or addiction. To mitigate these challenges and treat focal disabling pain conditions, the team is developing a novel strategy using gene therapy to silence pain-sensing neurons called nociceptors. The approach uses AAVs to overexpress potassium channels and builds on evidence that gain-of-function potassium channels can alleviate pain. The treatment strategy achieves dual precision using focal injections and nociceptor-specific promoters. Although the team is currently testing the technology in painful neuroma, it has the potential to be applicable to various other pain conditions, as well as therapies where cell-type precision in nociceptors is valuable.

Catheter directed delivery for cystic fibrosis

Gene therapy for treatment of pulmonary manifestations of cystic fibrosis (CF) have historically focused on inhaled delivery. Despite advances in inhaled delivery technology, CF gene therapy candidates have not been able to overcome the bronchial mucus barrier, which severely limits transduction. The team is developing a technique that uses targeted systemic arterial delivery to circumvent the mucus barrier and enable high expression of the therapeutic gene in airway basal cells. The technique involves catheter directed delivery of gene therapy candidates via bronchial artery. More specifically, the team will demonstrate proof of principle using porcine models and evaluate multiple viral vectors to determine the vector candidates best suited to transduce across endothelial cells.

Non-viral gene delivery and gene editing nanostructures for organ and cell-specific targeting

Lipid nanoparticle (LNP) structures are well-studied due to their ability to efficiently encapsulate mRNA, internalize cells, and release genetic cargo at the target site. Despite the accelerated rate of LNP development, LNP biodistribution and cell specificity have shown high tropism to the liver. Therefore, LNPs are less amenable for diseases associated with liver-off-site targets, such as the kidney, spleen, and brain. Addressing this need, the team aims to investigate and compare cellular uptake, internalization mechanisms, and biological responses of LNPs across various cell types. The strategy involves implementing industrial approaches for nanoparticle synthesis and identifying the optimal nanostructure for different cell types. Further characterization of intracellular fate of the LNPs will provide an understanding of their applicability in various therapeutics, such as cancer immunotherapy.

Gene editing for multisystem smooth muscle dysfunction syndrome (MSMDS)

 Hereditary rare genetic vascular disorders have a severe and predictable course that commonly cause stroke, MI, severe disability, and death in childhood. Among those, pathogenic missense variants in the ACTA2 gene, which encodes for a protein called alpha smooth muscle actin isotype 2, cause a severe syndrome called multisystem smooth muscle dysfunction syndrome (MSMDS). MSMDS is characterized by systemic smooth muscle cell (SMC) dysfunction, hypotension, aortic aneurysms, and devastating cerebrovascular disease that leads to death in the first three decades of life. As this disease recapitulates several features of common vascular disease, the team is currently leveraging a conditional knock-in mouse model of MSMDS to evaluate corrective genome editing strategies. With the goal of translating the innovation to patient-derived human arteries, the team will advance and validate the vector biology of a novel AAV that can target vascular wall cells to deliver a gene editing strategy for ACTA2.