FACIAL PAIN RESEARCH FOUNDATION 2019-2020 RESEARCH PROGRESS

Compiled and Submitted by Douglas K. Anderson, Ph.D., Eminent Scholar Professor and Chair Emeritus, Department of Neuroscience, University of Florida College of Medicine; Director of Research and Trustee, Facial Pain Research Foundation

Jerry Krbec MBA, FPRF Trustee

Michael Pasternak, Ph.D., FPRF Trustee

Trigeminal Neuralgia (TN), is a prime example of neuropathic pain which is pain caused by injury to or malfunctioning of the nervous system and is considered to be one of the most painful disease conditions known to humans.  In addition to the debilitating effects on the lives of those afflicted with neuropathic pain, the cost disorders like TN adds to the U.S. health care system exceeds $100 billion/year.  Yet despite the obvious escalating and pain fueled humanitarian and economic crises, funding targeted at discovering the basic molecular mechanisms underlying neuropathic pain conditions from traditional federal agencies, continues to be inadequate.  The meagerness of funding from these traditional sources has led the growing realization that discovering the root cause of and treatment for TN and other neuropathic pain conditions is going to have to be accomplished with funding from the private sector.  To this end, The Facial Pain Research Foundation (FPRF) was created a little over nine years ago in January 2011 to provide the critical elements that are necessary and sufficient to study these facial pain conditions.  Consequently, the sole mission of the FPRF is “…to establish a well-funded translational (i.e., fundamental discovery to clinical application) research continuum that is dedicated to identifying the mechanisms underlying neuropathic facial pain and to develop novel new therapeutic strategies that will permanently stop the pain of TN and related neuropathic pain syndromes”.  Since its inception, the FPRF has raised over $6,5 million dollars to support nine separate and distinct research projects.  The FPRF uses three fundamental criteria in choosing which proposals are to be considered for funding:  (1) the projects are novel, unique and perhaps even a little risky which I define as an “educated longshot” (2) the investigator(s) responsible for each project are among the leading researchers in their respective fields; and (3) there is significant diversification in the research strategies among the projects.  It is important to note that these FPRF funds have also served in a multiplier capacity in that some of our investigators have used FPRF support as seed funding to generate the necessary preliminary data which contributed to these investigators acquiring large grants from the NIH and other foundations and, in some cases, the acquisition of venture capital.  Total dollars from other sources that came to our consortium of investigators emanating, all or in part, from FPRF seed funds, is estimated to be somewhere over $50,000,000.

The purpose of this yearly update is to briefly highlight the progress made in selected projects in 2019 and to date in 2020.  Whereas in past reports I have attempted to present something about all funded projects irrespective of the progress made. However, the chaos and devastation caused by the highly infectious Covid-19 disrupted the substructure and organization of many sensitive non-vaccine development medical programs. Most of the labs conducting our research were closed for months due to the Covid crisis. Important data was lost, and all our programs were substantially delayed.  At my request, some of these investigators have described how Covid-19 impacted them causing their research to shut dow

   

The William H. and Leila A. Cilker Genetics Research Program to Find a Cure for Trigeminal Neuralgia (“Finding the Genes that Predispose to Trigeminal Neuralgia”)

Scott R Diehl, PhD, Department of Oral Biology, Rutgers University School of Dental Medicine

Kim J Burchiel, MD, Department of Neurosurgery, Oregon Health & Science University

Ze’ev Seltzer, PhD, University of Toronto Centre for the Study of Pain

An expanded Genome Wide Association Study (GWAS) has been completed using a substantially larger number of Trigeminal Neuralgia (TN) patients and a larger control group that included a wide diversity of race/ethnicity and geographic locations. GWAS studies detect associations with polymorphisms that are relatively common in the population (>5%). Since TN is very rare these cannot “cause” the condition to develop but may increase or decrease risk depending on which DNA variant an individual inherits. Results from this larger sample have not replicated previous findings that were based on smaller numbers of patients, nor have any new very strong signals located in other places in the genome emerged.

The study’s GWAS “failure to replicate” should not be considered a failure of the study. Some disorders such as autism have both effects of common polymorphisms found by GWAS approaches as well as rare variants that can only be found by DNA sequencing. Other disorders such as Spinocerebellar Ataxia are caused only by rare variants. The only way of knowing whether common polymorphisms studied by the GWAS method are important for TN was by rigorously performing the research and seeing the results. The study’s GWAS data now clearly indicate that TN does not have very strong genetic modifiers of risk that are common in of the population.

The finding that there are no common mutations responsible for TN, moved the research focus to examine potential uncommon mutations. As you will read below the Whole Genome Sequencing method yielded new data on a large number of very rare mutations present in TN patients.

One GWAS finding that remains of interest is a moderately strong association of a polymorphism adjacent to a gene involved in neuronal signal transduction and axon guidance. The polymorphism is highly correlated with the gene’s expression in neural tissues (i.e., how much of the protein coded by the gene is produced). This association in TN patients is highly relevant to ongoing studies by several labs focused on gene expression changes in neural tissues following nerve injury in animal models (including the study led by Allan Basbaum supported by the Foundation).

Since common polymorphisms studied by GWAS have at most a limited role in the etiology of TN, the alternative hypothesis that extremely rare protein-damaging mutations remains to be tested to see if this may be the primary cause of TN. Significant advances in pursuit of this aim have been achieved during the past year by Whole Genome Sequencing (WGS) of 101 TN patients. Analyses using advanced bioinformatics algorithms including artificial intelligence tools have revealed very rare protein-damaging mutations in 182 genes whose normal function is related to the nervous system and the biology of pain. Most of these mutations are found in the general population in only 1 person per 10,000 or less and some have never been seen previously in any human genome sequenced. This completely new information conclusively demonstrates that TN is not a single condition with a single cause. Instead, TN is a heterogeneous disorder with multiple different specific causes in different patients.

On average, each TN patient has inherited three different mutated genes, not just a single dysfunctional gene (as is the case in single gene disorders such as cystic fibrosis or Huntington’s disease). In this respect, TN is more similar in its genetic etiology to Parkinson’s disease, epilepsy, autism and other complex psychiatric and neurological disorders where each patient frequently inherits more than one mutated gene with each contributing substantially to risk of developing the condition.

Although 182 genes may seem a large number, when viewed from the perspective of their biological roles in neuronal tissues, the genes discovered actually fall into a much smaller number of biologically functional subgroups. Some patients carry mutations in one functional subgroup whereas others are affected by two or more such subgroups.

Based on lessons learned from genetic studies of other neurological disorders, it is likely that knowledge of the particular set of mutations each TN patient inherits can be used to guide decisions about which medications will be most beneficial while minimizing side effects. Development of a new diagnostic and prognostic screening panel for TN patients, using a comprehensive catalog of gene mutations, has now emerged as a feasible long-term translational goal emanating from this study and one of its most immediate clinical benefits. To assure that such a screening panel provides valuable clinical information for all TN patients, however, it is essential that all TN-causing mutations be included. This, in turn, requires DNA sequencing of many of the additional 900 TN patients who have already been recruited to the study’s cohort but not yet sequenced.

When these exciting results are reported to the scientific community at conferences and in peer-reviewed publications, the study’s discovery of very rare mutations causing TN will motivate academic and pharma/biotech research groups to give these genes a close look for development of more effective analgesics, gene therapies and other innovative approaches leading to the ultimate goal of finding cures for every patient affected by TN.

 

Mapping Toward a Cure: Identification of Neurophysiological Signatures of Trigeminal Neuralgia (TN) Pain

A University of Florida (UF) team consisting of John K, Neubert, Ph.D. (Project Leader), Mingzhou Ding, Ph.D., Robert Caudle, Ph.D., Marcelo Febo, Ph.D., and Todd Golde, M.D., Ph.D. are investigating the neurophysiological signature of trigeminal neuralgia (TN) pain and developing promising new therapies. This group represents expertise in facial pain, structural and functional neuroimaging, neurophysiology, molecular biology, pharmacology, and viral vectors.  Initially, this project was designed to include both preclinical animal models of trigeminal nerve pain and human studies using imaging studies from TN subjects to investigate the cause of TN. The preclinical animal studies have yielded the novel therapeutic agent, (CGRP-Botox (see Dr. Caudle’s separate report). Dr. Febo has provided additional animal imaging analyses and has identified unique connections within the brain following trigeminal nerve injury. These findings confirm the belief that unique pathways relevant to trigeminal nerve pain exist and can provide a foundation for studying humans using the same imaging analyses. The human studies have found critical brain regions important in transmitting pain in TN patients and we identified an exciting relationship relating to neuroinflammation, chronicity, and surgery. These findings provide the foundation for a journal article that is currently in preparation and will be submitted in the next few months. We have made significant progress towards completing the goals of this project and will expand on these exciting results to provide a pathway for finding a cure for TN.

 

HUMAN STUDIES

Recruitment

Control TN with surgery TN without surgery TN1 TN2 TN1/TN2
3 20 22 28 3  11

Supported by the Facial Pain Research Foundation (FPRF). we have undertaken the task to image the brain of TN patients using a multimodal approach (25 females} (TN1, TN2, TN1/2) enrolled in our study). Subsequently, these patients received a clinical examination and then underwent functional and structural magnetic resonance imaging (MRI) at the McKnight Brain Institute of the University of Florida. (MBI). Functional imaging, functional MRI (fMRI) data were acquired while the patient rated their momentary pain levels using a tracking ball. During structural imaging, diffusion MRI data were acquired while the patients rested inside the MRI scanner. Subject recruitment has ended, and we are working hard to complete completing the analysis on this final group.

 

Brain Activations during Trigeminal Pain      

We recorded pain ratings during the fMRI scans to allow us to uncover brain regions that become activated during a TN pain episode. There are several interesting regions that are activated including the anterior insula, caudate, putamen, thalamus, and prefrontal cortex. This is important because identification of such brain regions serves as a fundamental and necessary step towards finding central-nervous system targets for effective therapeutic interventions in TN.

 

Neuroinflammation and TN

The pontine crossing tract (PCT) is a region of the brain that links the brainstem and left/right cerebellum and is an area of interest for processing trigeminal pain. We evaluated a free water (FW) analysis of this region as a marker for TN-related neuroinflammation. We have shown there is a significant positive correlation between free water in the PCT and disease duration in non-surgery TN1 patients. As a comparison, we analyzed scans from subjects with temporomandibular joint disease (TMD), another chronic orofacial pain condition. We found that the increase in FW in the PCT did not correlate with disease duration in the TMD cohort.  This finding is important because it indicates that there may be a specific mechanism or biomarker for TN, which is not present in other chronic orofacial pain disorders. These findings suggest that in non-surgery TN1, as the disease progresses, neuroinflammation becomes more severe; this pattern is specific to TN1 and not found in TMD. This is important because the FW analysis may be used as both a diagnostic biomarker of TN and to study mechanisms related to TN.

 

Novel regions related to TN pain:  Central Ignition via Thalamic Disinhibition

For the six TN1 patients who exhibited clear on-off pain attack patterns in the scanner, we found that specific regions of the brain (thalamus and anterior cingulate cortex) affected the pain onset. Immediately prior to the onset of pain attacks (Pre-pain), top-down inhibitory influence from anterior cingulate cortex (ACC) to thalamic, ACCàThalamus, was reduced relative to a baseline condition (Resting), causing thalamic disinhibition. Concurrently, the thalamic input into ACC, an important structure of the pain matrix, was increased immediately preceding the onset of pain attacks, a phenomenon we referred to as central ignition. These results outline a clear mechanism of TN pain and suggest a possible therapeutic approach: by enhancing the ACC top-down influence we may restore the inhibitory control over the thalamus and thereby reduce the level of pain and the frequency of pain attacks.

 

FUTURE STUDIES

We plan to recruit and scan 15-20 more TN1 and control subjects. Dr. Febo’s group will continue to analyze the animal imaging data set. These results will be included in the preclinical animal imaging manuscript (in preparation)

 

Exploring Neuropeptide Guided Botulinum Light Chain for use in Blocking Pain Transmission 

Robert M. Caudle, Ph.D., Department of Oral and Maxillofacial Surgery, University of Florida

Progress Report

The goal of this project is to develop and evaluate a novel conjugate of calcitonin gene related peptide and the light chain of botulinum toxin A (CGRP-BotLC) as a therapy for trigeminal neuralgia. This conjugate replaces the heavy chain of botulinum toxin, which binds to motor neurons. The CGRP will bind primarily to sensory neurons. The theory behind the use of this agent is that CGRP will bind to G-protein coupled CGRP receptors on nociceptive neurons of the trigeminal ganglia. The receptors will then transport CGRP-BotLC into the cells where the light chain of botulinum toxin will disable a protein critical for neurotransmitter release. Without this protein the neurons will not be able to transmit pain signals upstream to the next neuron, thus blocking the pain from trigeminal neuralgia. Wildtype botulinum toxin, as in Botox®, can inhibit neurotransmission in motor neurons for up to 6 months; thus, it is expected that a single injection of CGRP-BotLC will provide pain control for several months. Since botulinum toxin only has an effect when it gets internalized by neurons CGRP-BotLC will have no effect on neurons that lack CGRP receptors such as motor neurons. This modification will provide greater safety and efficacy than currently available clinically used botulinum toxins. To evaluate this conjugate three specific aims were proposed.

 

Specific Aim 1 – Scale up production of CGRP conjugated botulinum A light chain (CGRP-BotLC) for in vivo studies.

Specific Aim 2 – Determine appropriate dose and route of administration for CGRP-BotLC to effectively control nociception in rodent models of neuropathic pain.

Specific Aim 3 – Perform studies to determine how long the conjugate blocks pain.

 

Specific Aim 1 – We have successfully generated the CGRP-BotLC conjugate. The activity of 1mg of the total synthesized agent was confirmed by assay. The remaining 6mg will be tested for activity within the next couple of weeks. It is expected that the remaining 6 mg will be sufficient for the behavioral assays proposed

Specific Aim 2 – An initial experiment was conducted with CGRP-BotLC in 9 rats to gauge the utility of a nerve injury model for evaluating the agent and to estimate the dosing range. The animals were trained in Orofacial Pain Assessment Devices (OPAD) using mechanical stimuli.  Testing in the OPADs was continued for another 30 days after nerve injury. The infraorbital nerve injury produced a large decrease in licking events (feeding) in the OPAD assay indicating that the animals were more sensitive to the mechanical stimulus after the injury than prior to the surgery. Following the injections, the CGRP-BotLC animals returned to baseline levels of licking within a few days whereas the PBS treated animals remained hypersensitive to the stimulus for several weeks. These findings were compelling; however, due to the limited number of rats in this pilot study the two groups did not differ statistically.

Specific Aim 3 – The data demonstrates that the rat infraorbital nerve injury model does not produce hypersensitivity long enough to evaluate the full duration of CGRP-BotLC. When work in the laboratories can resume a new larger group of animals will be tested using a longer lasting nerve injury model.

 

Interpretation

The data from this first pilot experiment provides several interesting observations. The first is that CGRP-BotLC is substantially safer than wildtype botulinum toxins such as Botox®.  This improvement in safety was expected due to the removal of the heavy chain from botulinum toxin. The heavy chain targets the protein to motor neurons, which can lead to suppression of respiration and death. The second observation  is that a dermal injection of CGRP-BotLC into the site where hypersensitivity is measured following the nerve injury reverses the hypersensitivity. Normal baseline activity in the OPAD was observed within a couple of days following CGRP-BotLC injection. This reversal is maintained throughout the remainder of the experiment. The vehicle treated animals, on the other hand, recovered spontaneously 22 days following the injections. These findings indicate that CGRP-BotLC has a long duration of action. Unfortunately, the nerve injury model did not provide a long enough period of hypersensitivity to evaluate the full duration of action of CGRP-BotLC. Finally, the localized dermal injection appeared to be highly effective at reversing hypersensitivity. This represents the ideal route of administration for treatment of trigeminal neuralgia because it will require less CGRP-BotLC than intravenous administration and the dermal route will limit uptake by non-targeted cells that express CGRP receptors.

 

Investigating Protein-Lipid Interactions in Peripheral Nerve Myelin

Lucia Notterpek, Ph.D.Professor and Associate Dean of Biomedical Research, University of Nevada Reno School of Medicine

Study staff: Ye Zhou, MS, graduate student

Progress report: May 2019-April 2020

 

Project 1: Cholesterol homeostasis in peripheral nerve myelin

We continued to make excellent progress on this project, even though Ye Zhou, the graduate student leading the work, left the lab in September 2019. She is now a postdoctoral fellow at Regeneron Pharmaceuticals, in New York. From Ye’s work, we have published 3 manuscripts so far—with 2 articles examining the basic biology of lipid and cholesterol metabolism in myelin-forming cells of peripheral nerves, and one article on dietary intervention (these publications are listed below, and each acknowledges FPRF for supporting the work).

Through Ye’s work we learned that when cholesterol is not in the correct location within the glial cells, myelin is abnormal and, therefore, does not properly insulate the nerve. The manuscripts lead by Ye provide new knowledge for the field and validate lipid metabolism as a target pathway for neurological disorders with myelin defects.

The article on dietary intervention opened a new area of investigation for us. In agreement with existing literature, we found that a lipid-enriched, high fat diet (HFD) benefited nerve myelination and alleviated myelin defects in genetic models of neuropathies. After the HFD feeding, nerves from neuropathic mice displayed reductions in hallmarks of myelination defects, including aberrant Schwann cell mitosis and de-differentiation. Nerves from neuropathic mice on HFD also exhibited decreased macrophage infiltration with a normalized count of CD11b+ macrophages, indicating reduced inflammation. However, the diet we tested in the mice is not healthy for humans, thus, based on discussions with a human nutritionist (Dr. Percival at UF), we have shifted our work to developing a healthy lipid-enriched diet formulation, which is based on extra virgin olive oil (EVOO).

In our first pilot study, the EVOO was a single olive species product (Arbosana), purchased from a local orchard (Alachua, Florida), and subsequently analyzed by gas chromatography for fatty acids composition, and by HPLC for polyphenol composition. In addition to the EVOO providing 35% of total calories in the diet (high fat) we added phosphatidylcholine, which was shown in a recent high-profile publication to benefit myelin repair (Fledrich et al., Nature Communications 2018).

In total we have tested 88 mice on this diet formulation, with the mice representative of varying severity of myelin defects. Unfortunately, the study was interrupted by the COVID-19 pandemic, and some of the animals had to be collected before the full course of the intervention, which was set at 8 weeks long. We have all the nerve samples collected from these mice, but they have yet to be processed for analyses.

During this study, and with the pandemic halting all hands-on experiments, I have designed and requested two additional test diet formulations:

  • Same 35% fat content from EVOO but using two 2 different olive species (50% Arbosana and 50% Koroneiki) which provides higher polyphenol content, per our HPLC data. This formulation also has the added choline, as in the initial formulation.
  • Same 35% fat from EVOO (Single species Arbosana), with added Phosphatidylcholine, and increased D3, B12, and Biotin. In independent studies, each of these vitamins have been shown to benefit neuropathic symptoms, including myelin defects and pain.

These two new custom diets are now being made and will be shipped to my new lab in Reno, Nevada.

Project 2: Cholesterol homeostasis in peripheral nerve myelin, with a focus on statins

As previously stated, Mohammed Al Salihi, MD, PhD, postdoctoral fellow, was the key investigator on this project, but he resigned in March 2019, and returned to training in medicine. While my students repeated some of his results with statins, the study must be taken up by a more experienced scientist. Nonetheless, the additional data from the last year continues to show statins have a very strong negative influence on myelinating glial cells—a finding that will be repeated and further analyzed for publication (this will be a priority once my lab is open in Reno).

Publications from the last 12 months:

  1. Zhou Y, Lee S, Bazick, H, Miles J, Tavori H, Landreth GE, Fazio S, Notterpek L. PMP22 regulates cholesterol trafficking and ABCA1-mediated cholesterol efflux. J Neuroscience, 2019, 39(27):5404-5418.
  2. Zhou Y, Bazick, H, Miles J, Fethiere A, AISalihi M, Tavori H, Fazio S, Notterpek L. “A neutral lipid-enriched diet improves myelination and alleviates peripheral nerve pathology in neuropathic mice. Experimental Neurology, 2019 Nov 321:113031
  3. Zhou Y, Borchelt D, Bauson JC; Fazio S, Miles JR, Tavori H, and Notterpek L. Subcellular diversion of cholesterol by gain- and loss-of-function mutations in PMP22. 2020, Glia, in press

In summary, even with the COVID situation, I was able to pack up my lab and move it to Nevada. Lab equipment, cold and frozen samples have all arrived, and one of the neuropathic mouse lines have been shipped. Hopefully, the line for the compression-induced neuropathy will be shipped next week.

 

Novel sensory target genes for novel trigeminal neuralgia pharmacotherapies

Allan Basbaum, Ph. D, FRSProfessor and Chair- Department of Anatomy, University of California, SanFrancisco

Even though we have not been able to keep the laboratory open for the last two months, we were quite productive before the shutdown. We have completed the in situ hybridization analysis of the expression of several of the so-called “dark” genes in sensory neurons of the dorsal root ganglion (DRG), before and 7 days after unilateral peripheral nerve injury (spared nerve injury; SNI). Of particular interest are our studies of the Cacna2d voltage-dependent calcium channels, which include 4 alpha subunits (2d2, 2d3 and 2d4). In these studies, compared the distribution and expression levels of the dark Cacna2d subunits to those of the well-characterized 2d1 subunit, which has been implicated in many neuropathic pain conditions and is the target of gabapentin and pregabalin. And most recently we evaluated three potassium voltage-gated channel family A members (b2, b3 and b6). The latter are of interest as there is good reason to believe that a decrease in potassium channel expression is associated with hyperactivity, indeed spontaneous activity, of neurons in general and in sensory neurons in particular. These particular Kcnab subunits modulate the activity of the parent channel.

Using multiple labeling in situ hybridization we determined that whereas Cacna2d1 is expressed in a large and mixed population of DRG neurons (both myelinated and unmyelinated), 2d2 and 2d3 are mostly expressed in non-overlapping subsets. Of particular interest is our finding that 2d4, which is barely detectable under normal conditions, is strongly induced in a mixed population of DRG neurons. To what extent this subunit contributes to ongoing neuropathic pain will require generating mice in which the gene is deleted from sensory neurons. That is on the list of experiments to begin when we return to the laboratory. We found that Kcnab2, Kcnab3 and Kcna6 are expressed at variable levels in a mixed population of sensory neurons, with Kcnab2 expressed at much higher levels (~30x) and overlapping with both b3 and b6. As of this time, we did not detect significant changes in expression after injury.

We are continuing this analysis and are extending it to CNS neurons (spinal cord and trigeminal nucleus caudalis). We are also examining the effects of inflammation on expression levels of the channels.

Evaluation of adeno-associated virus (AAV) constructs directed at pain pathway targets – a rapid approach for identification of effective therapies for trigeminal neuralgia

Todd E. Golde, M.D., Ph.D.

Yona Levites, Ph.D. 

Robert M. Caudle, Ph.D. 

John K. Neubert, D.D.S., Ph.D. 

University of Florida

The primary objective of this project is to design and synthesize a number of viral vectors directed at specific genes related to pain processing and then determine which of these constructs are safe and the most efficacious, doing so initially in animals.  Dr Golde’s team will utilize established expertise in Adeno Associated Virus (AAV) delivery systems to evaluate the effectiveness of these vectors for reducing pain.

 In short, gene therapy or delivery is the use of a viral construct to put in a copy of a gene that replaces a missing or defective gene; overproduces a protein that promotes cell growth and/or is “protective”, e.g., can control pain; and bioengineer stem cells and other types of cells to produce missing or protective proteins.

In the first stage several AAV vectors were created and evaluated.

The next stage included cloning potential pain modifier genes into the virus for delivery to rat’s sciatic nerve. Among genes tested are four potassium channels, six opioid receptors and secreted endogenous opioid neuropeptide and peptide hormone beta-endorphins.

Currently major technical obstacles have arisen with packaging these genes into the virus, due to their effects on the cells.

We have taken a multipronged approach to overcome some obstacles that have arisen during our studies. First, we have had challenges with reproducibly achieving robust expression of the virally encoded transgene in peripheral nerves of the mice. With help from Dr. Edgardo Rodriguez, we have now achieved more robust transduction of the mouse trigeminal nerve. This was accomplished by i) altering the methodology for injection to directly inject the whisker pad and ii) screening multiple different viral capsids (these coat the viral vector and determine its entry into cells) to identify those capsids that enable robust transduction. Second, we have encountered unexpected challenges in generating the viral vectors that encode leak channels designed to block pain signaling.  Expression of these leak channels was our most direct way to test our gene therapy approach. The reason this issue was unexpected in that we have created hundreds of viral vectors, and never encountered this problem. We have had instances where the first attempt to make virus failed, but standard trouble shooting enabled generation of viral vectors that were of sufficient quality for the experiments to work. In this case, those initial trouble shooting efforts, which were repeated at least three times, failed. At that point we suspected that the expression of the leak channel during the generation of the virus was causing problems resulting in low yields. We thus reengineered the leak channel vectors, so that we could monitor expression directly and ii) limit expression during vector generation. These efforts have been partially successful enabling us to produce virus with enough yields to begin studies in mice. Viral yields are still not optimal, and we have several additional steps that we are taking to see if they can be improved. Unfortunately, our progress was halted when UF laboratories were almost completely shut down by the COVID-19 pandemic. This shutdown meant that no new experimentation was permitted. As of early June, we are now able to ramp up research efforts, but we are still severely restricted on what we can do regarding animal studies. We will begin additional animal studies needed for this project, as soon as we are able, but at this point we do not yet know when we will be able to conduct these studies. As we being to resume some activities in the lab, we will continue our efforts to optimize production of our vectors continuing the functional leak channels. We are also generating novel cargoes that are designed to knockdown expression of key pain signaling proteins, specifically trying to silence targets that are genetically validated. In this case rather than artificially overexpressing a protein, we are simply trying to repress an endogenous protein that is responsible for mediating the sensation of pain. As no protein is made during the viral vector production, we do not anticipate problems that we enc