Category Archives: Genetic Therapy

Researchers at the Salk Institute have made a breakthrough that could lead to new treatments for genetic hearing loss. Gene therapy that delivers a particular protein can ensure faulty hair cells grow correctly, allowing for improved hearing.

Sensory hair cells are a vital part of our auditory system. They line the surface of the cochlea with long structures called stereocilia, which vibrate in response to sound waves and produce electrical signals that are then sent to the brain. But one form of genetic deafness arises due to a lack of a protein called EPS8, which regulates the length of these hair cells. Without it, theyre too short to work properly.

For the new study, the researchers investigated whether restoring EPS8 could help those hair cells grow to their normal length and improve hearing. Working in mice that had been engineered to lack EPS8 and, as such, were deaf, the team then experimented with using an adeno-associated virus as a vehicle to deliver the protein to the animals inner ears.

And sure enough, they found that the added EPS8 made the stereocilia grow longer, restoring some function to the cells that pick up lower frequency sounds.

Salk Institute/Waitt Advanced Biophotonics Core

However, there are some caveats. The treatment didnt work in mice after a certain age, suggesting its important to get in early before the hair cells mature. In humans, that would require applying the gene therapy in utero, since by birth it would already be too late. But the team hopes that with further study, that window for treatment could be widened.

EPS8 is a protein with many different functions, and we still have a lot more to uncover about it, said Uri Manor, co-senior author of the study. I am committed to continuing to study hearing loss and am optimistic that our work can help lead to gene therapies that restore hearing.

Other teams have found promise in restoring hearing through gene therapy by targeting other genes. That includes regrowing either inner or outer hair cells, correcting mutations that cause them to become disorganized, or repairing age-related damage to other structures.

The new research was published in the journal Molecular Therapy Methods & Clinical Development.

Source: Salk Institute

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New gene therapy could prevent genetic hearing loss - New Atlas

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Homology Medicines Announces Optimized, In Vivo Gene Therapy Candidate for the Treatment of ... - El Paso Inc.

SAN FRANCISCO, Aug. 11, 2022 /PRNewswire/ -- The global gene therapy market size is expected to reach USD 10.0 billion by 2028 registering a CAGR of 20.4% during the forecast period, according to a new report by Grand View Research, Inc. The market growth is attributed to the increasing prevalence of cancer coupled with the lack of effective treatment for the disease. Constant expansion of cancer genetic studies has deciphered relevant information about cancer-related molecular signatures.

Key Industry Insights & Findings from the report:

Read 180 page full market research report for more Insights, "Gene Therapy Market Size, Share & Trends Analysis Report By Indication (Large B-cell Lymphoma, Beta-Thalassemia Major/SCD), By Vector Type (Lentivirus, AAV), By Region, And Segment Forecasts, 2021 - 2028", published by Grand View Research.

Gene Therapy Market Growth & Trends

This has driven the clinical trials for advanced cancer therapeutics, thereby driving the market growth. In addition, expansion of this mode of treatment in non-cancer applications with approval of therapies, such as approval of Bluebird Bio's Zynteglo in June 2019 for -thalassemia and others, demonstrates the shift in the preferences of companies towards other unsaturated segments.

Various universities and institutes are observed to exhibit a broad portfolio in the pipeline, which is anticipated to boost revenue generation in the future. The market is anticipated to witnesses a short-term revenue slowdown due to a shift in focus and delay in product launches. However, initiatives for developing novel trial designs, improving the regulatory environment, and managing the supply chain are expected to play a critical role in minimizing the impact of the current global crisis.

Gene Therapy Market Segmentation

Grand View Research has segmented the global gene therapy market on the basis of indication, vector type, and region:

Gene Therapy Market - Indication Outlook (Revenue, USD Million, 2017 - 2028)

Gene Therapy Market - Vector Type Outlook (Revenue, USD Million, 2017 - 2028)

Gene Therapy Market - Regional Outlook (Revenue, USD Million, 2017 - 2028)

List of Key Players of Gene Therapy Market

Check out more related studies published by Grand View Research:

Browse through Grand View Research's Biotechnology Industry Research Reports.

About Grand View Research

Grand View Research, U.S.-based market research and consulting company, provides syndicated as well as customized research reports and consulting services. Registered in California and headquartered in San Francisco, the company comprises over 425 analysts and consultants, adding more than 1200 market research reports to its vast database each year. These reports offer in-depth analysis on 46 industries across 25 major countries worldwide. With the help of an interactive market intelligence platform, Grand View Research Helps Fortune 500 companies and renowned academic institutes understand the global and regional business environment and gauge the opportunities that lie ahead.

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Gene Therapy Market to be Worth $10.0 Billion by 2028 with CAGR of 20.4%: Grand View Research, Inc. - Benzinga

bluebird bio has reported its financial results and business highlights for the second quarter of 2022, in which the company revealed the anticipated launch of both betibeglogene autotemcel (beti-cel) and elivaldogene autotemcel (eli-cel) gene therapies in the fourth quarter of the year.

The news comes after both therapies were endorsed by the US Food and Drug Administration (FDA) Cell Tissue & Gene Therapy Advisory Committee (CTGTAC) in June 2022.

Beti-cel is under priority review for the treatment of people with transfusion-dependent beta-thalassaemia (beta-thal), a rare genetic blood disease caused by a gene defect that impairs the ability of red blood cells to produce haemoglobin. Patients with the most severe form of beta-thal develop life-threatening anaemia and have to undergo regular blood transfusions, a lengthy process typically needed every two to five weeks.

Eli-cel is under priority review for the treatment of early active cerebral adrenoleukodystrophy (CALD) in patients under the age of 18 who do not have an available and willing matched sibling donor. CALD is a rare neurodegenerative disease that primarily affects young children and leads to irreversible loss of neurologic function and death.

Beti-cel and eli-cel have Prescription Drug User Act Fee (PDUFA) goal dates of 19 August 2022 and 16 September 2022, respectively. If approved, the company anticipates that both therapies will be availabile in the fourth quarter of 2022.

Andrew Obenshain, chief executive officer, bluebird bio, said: The second quarter marked significant progress for bluebird bio and a precedent-setting moment for the field of gene therapy.

With the FDA advisory committees unanimous support for beti-cel and eli-cel for their target indications, we are now laser-focused on commercial readiness and, if approved, we anticipate launching both therapies in the fourth quarter of this year.

bluebird bio also reported that it remains on track to submit a Biologics Licensing Application (BLA) to the FDA for lovotibeglogene autotemcel (lovo-cel) for sickle cell disease in the first quarter of 2023.

The company reported that it ended the quarter with $218m in restricted cash, cash and cash equivalents and marketable securities, having raised approximated $24.7m in gross proceeds through its At-the-Market (ATM) equity facility.

The company is exploring additional financing opportunities, including public or private equity financings and monetising any priority review vouchers that may be issued upon approval of beti-cel or eli-cel.

Excerpt from:
bluebird bio reveals plans to launch two gene therapies - PMLiVE

Instead of trying to mop up disease-causing proteins once they have been produced, like most small molecule or antibody therapies, gene-silencing treatments target messenger RNA (mRNA) to cut off production of these proteins at source. And pioneering pharmaceutical companies working on this technology now have neurodegenerative diseases in their sights.

Neurodegenerative diseases such as Alzheimers, Parkinsons and Huntingtons are common and most lack disease-modifying therapies or cures.

At least 1 in 14 of the UK population aged over 65 years has dementia, with 6070% of those cases classed as Alzheimers disease[1,2]. Data also show that 1 in 37 people in the UK will be diagnosed with Parkinsons disease in their lifetime, and 1 in 10,000 will develop Huntingtons disease[3,4].

In December 2021, Ionis Pharmaceuticals, based in Carlsbad, California, in partnership with Biogen, based in Cambridge, Massachusetts, published data from the first trial of a gene-silencing drug BIIB080 for mild Alzheimers disease, showing it to reduce key Alzheimers biomarkers and cause no serious adverse events[5].

However, the publication of the Ionis trial came on the heels of two high-profile gene-silencing drug failures for Huntingtons disease, leaving those affected by the condition despondent. Understanding these failures may go some way to improving the next generation of gene-silencing drugs.

There are molecules that are in the wings, that are longer lasting, safer and more potent, says chemist Jonathan Watts, associate professor at the RNA Therapeutics Institute at the University of Massachusetts Medical School.

Gene-silencing therapies act to stop the translation of mRNA, providing a method to remove the harmful proteins formed from mutated genes that can lead to disease. The most established method uses antisense oligonucleotides (ASO) short synthetic single strands of DNA and RNA able to knock down genetic expression through base-pairing with their corresponding sense strands of mRNA, before they can be translated into proteins (see Figure).

Since 2017, ASOs to treat spinal muscular atrophy (nusinersen [Spinraza; Ionis Pharmaceuticals]) and Duchennes muscular dystrophy (casimersen [Amondys 45; Sarepta Therapeutics]) have led the way. Part of the attraction is the simplicity of designing high affinity drugs this way.

Everybody can match As with Us and Cs with Ts, jokes Watts.

ASOs can simply block access to the RNA base sequence to stop translation, but a new generation of ASOs, known as Gapmers, are exploiting another mechanism. They are made up of short DNA sequences with RNA-like segments on each side to provide higher affinity to the target, plus resistance to degradation by nucleases. When they bind to mRNA transcripts they are able to recruit an enzyme called Ribonuclease H, which will trigger the RNAs destruction (see Figure).

Ionis Pharmaceuticals Alzheimers ASO BIIB080 was designed to lower levels of tau protein, which when misfolded is thought to cause the neurofibrillary plaques found in patients brains. In the phase I trial, launched in 2017, it was administered intrathecally to 44 patients aged 5074 years with mild Alzheimers disease over three months and showed an ability to reduce tau and associated protein markers in the cerebrospinal fluid by 3050%[5].

The impact on cognition will not be clear until results of the phase II trial, which is set to start in summer 2022, with an estimated completion date of December 2026[6].

Ionis is also developing an ASO drug for Parkinsons disease ION464 designed to prevent the production of the alpha-synuclein protein, known to accumulate in the brains of people with the condition, and has licensed another tofersen, for a rare, genetic form of amyotrophic lateral sclerosis (ALS) called superoxide dismutase 1 (SOD1) ALS to Biogen.

In July 2022, the US Food and Drug Administration (FDA) approved a new drug application and accelerated approval pathway for tofersen, despite the drug failing to meet the primary end point in a phase III trial in 2021[7]. Integrated 12-month data from two trials included in the FDA filing suggest that individuals who start tofersen earlier experience a slower rate of decline.

The promise of this gene-silencing approach has been tempered by the failures of two ASO drugs to treat Huntingtons disease a rare genetic condition that causes the progressive degeneration of nerve cells in the brain.

One drug, manufactured by Massachusetts-based Wave Life Sciences, was designed to target the mutant Huntingtons gene (HTT) by recognising a single nucleotide polymorphism (an approach that only works in a subset of patients). However, in an early phase Ib/IIa trial, the drug failed to significantly lower levels of the mutant protein in the cerebrospinal fluid[8].

Roches ASO tominersen (licensed from Ionis Pharmaceuticals in 2017) was designed to reduce both the mutant and healthy versions of the protein, but even though a previous phase I/II trial did show lower levels of the protein in treated patients, it failed in its phase III trial of 800 patients in 2021, producing worse outcomes, with five cases of hydrocephalus a build-up of fluid on the brain in patients receiving the highest dosages of 120mg[9,10].

Does this indicate a fundamental failure with the approach? The answers are a definitive no, says Edward Wild, professor of neurology and consultant neurologist at the National Hospital for Neurology and Neurosurgery, Queen Square, and associate director of University College Londons Huntingtons Disease Centre, who was involved in the tominersen trial.

What both failures show in different ways is how difficult it is to successfully deliver ASOs to the brain. Wild says Wave Life Sciences drug failed to engage the target. Thats really a question of going back to the chemistry of the ASOs and making them better.

Tominersen, however, did reach its target, but the high doses given to facilitate delivery deep into the basal ganglia caused toxicity, triggering an inflammatory response in the brain.

I think the doses that were chosen were too high; in retrospect, we would do it differently

I think the doses that were chosen were too high, says Wild. In retrospect, we would do it differently.

In subsequent analysis, Roche found that those patients at an earlier stage of the disease, or who were younger, did better than the group as a whole, suggesting they were more able to tolerate the high ASO doses and gain a benefit[11]. As a result, Roche plans to repeat a dose-ranging trial in this patient sub-group to try and test that hypothesis.

The failed drugs were based on chemistry developed in the 1980s and, according to Matthew Wood, professor of neuroscience in the Department of Paediatricsat the University of Oxford, there are many new chemical improvements that have come along in the past decade.

The brain is protected by the bloodbrain barrier that prevents entry to most molecules. For this reason, current ASOs are injected directly into the spinal fluid, which provides better access to the brain but makes frequent administration difficult.

A second generation of ASOs include chemical modifications, with the non-bridging oxygens of the oligonucleotide phosphate backbone replaced with sulphur atoms (known as phosphorothioate modification). This improves their ability to resist attack by nucleases and also enhances protein binding and cell uptake properties, as well as overall bioavailability. So, although these drugs will still need to be administered intrathecally, a lower dose is likely to be effective, providing an improved therapeutic window.

The other approach to gene silencing takes advantage of RNA interference, one of the cells natural defenses against viral RNA to prevent it being translated into proteins. It works using a multi-protein assembly called RNA-induced silencing complex (RISC), which is able to break down double-stranded RNA to prevent translation. The complex loads one of the strands of an RNA molecule (the anti-sense strand) and here it acts as a template. Any complementary RNA transcripts in the cell will be destroyed before a protein can be formed from them (see Figure).

This can be exploited therapeutically if small interfering RNA (siRNA) molecules are delivered to cells. Here, they will automatically be loaded into RISC and the anti-sense strand will stay there, bound for months, continuing to remove any complementary mRNA produced by the cell. This means continued dosing is not needed but getting the siRNA into the cell in the first place is even more difficult than for an ASO, owing to its larger size and its negatively charged surface.

If you inject a single stranded ASO into the bloodstream, it is taken up pretty effectively but a duplex RNA, something thats fully double stranded, does not show much cell uptake, explains Watts.

siRNAs are probably of the order of somewhere between one and ten-fold more potent than an ASO system, in theory

siRNAs ability to silence a gene is much greater though, probably of the order of somewhere between one and ten-fold more potent than an ASO system, in theory, says Wood, so there is a strong incentive to develop better delivery mechanisms.

In practice, resolving the chemistry and optimising the delivery has just taken longer to achieve [than for ASOs].

Alnylam Pharmaceuticals has pioneered the siRNA approach and, in 2021, it submitted a clinical trial application in collaboration with Regeneron Pharma to the Medicines and Healthcare products Regulatory Agency for a phase I study of ALN-APP, an siRNA to treat early-onset Alzheimers disease by silencing the amyloid precursor protein (APP). The siRNA is designed to be delivered intrathecally in a single dose.

This will be Alnylams first siRNA programme using its new C16 conjugation technology to improve delivery to the brain. In addition to several modifications to the oligonucleotide backbone, the siRNA molecule is conjugated to a hexadecyl group. Exactly how this improves uptake in the brain is still being investigated.

We hypothesise that it promotes moderate affinity interactions with the extracellular matrix and various cell membrane components, thus allowing for binding and uptake into various cell types within the brain as well as the lung and the eye, explains Vasant Jadhav, head of the RNAi Platform at Alnylam Pharmaceuticals.

The company, based in Cambridge, Massachusetts, intends to use its promising siRNA approach to treat other neurodegenerative diseases.

There are a number of genetically defined neurodegenerative diseases where there is significant potential for CNS-targeted RNAi therapeutics to have an impact,

We have development candidates that are undergoing preclinical evaluation for the potential treatment of Huntingtons disease and ALS in collaboration with Regeneron. We certainly feel that there are a number of genetically defined neurodegenerative diseases where there is significant potential for CNS-targeted RNAi therapeutics to have an impact, says Jadhav.

Now a third generation of gene-silencing drugs for delivery to the CNS are making their way to the clinic. A novel design comes from the research of Anastasia Khvorov at the University of Massachusetts and is now being developed through start-up company, Atalanta Therapeutics, also based in Massachusetts. They are developing a new chemically modified siRNA architecture called divalent siRNA (di-siRNA), which is made up of two parallel modified oligonucleotides linked together[12].

di-siRNA may be an improvement over both ASO and over ordinary siRNA, in terms of distribution, durability and tolerability

So far, according to Alicia Secor, chief executive of Atalanta Therapeutics, their performance suggests that di-siRNA may be an improvement over both ASO and over ordinary siRNA, in terms of distribution, durability and tolerability.

Atalanta is collaborating with Biogen on an HTT-targeting treatment for Huntingtons disease, and with Genentech, based in San Francisco, California, to develop di-siRNA therapeutics for neurodegenerative diseases, including targets associated with Parkinsons and Alzheimers, plus developing its own pipeline.

Another third-generation approach comes from start-up Ceptur Therapeutics, based in New Jersey, which has attached U1 adapters to siRNA molecules. These short single strands of artificial RNA bind to the protein U1 snRNP part of the machinery that modifies RNA molecules inside the nucleus after they are made. In this case, the adapters prevent the addition of the polyA tail stretch of the mRNA molecule, which is needed for translation without it the nascent RNA is degraded. Ceptur recently completed a US$75m financing round to continue developing its therapeutic for Huntingtons disease.

Gene-silencing therapies based on siRNA are also now being delivered as gene therapies, using an adeno-associated viral vector to deliver the gene that makes the therapeutic RNA molecule.

Dutch biotech company, uniQure biopharma, is working on this approach to treat Huntingtons disease with its gene therapy candidate, AMT-130. The therapy delivers the gene directly to the caudate and putamen two deep brain structures that are affected in people with Huntingtons disease, where information is processed. This will then continually produce the engineered RNA molecule, which will bind any mutated HTT protein and mark it for destruction. The procedure is done by catheter, guided by MRI. The first two patients in Europe have been dosed in uniQures phase I/II clinical trial, which is underway in Poland and is currently expanding to the UK[13]. Its a pretty invasive approach, high risk, [but] high reward, says Wild, who is part of the University College London and Cardiff clinical trials team.

Ultimately the ambition of many of those developing drugs for neurodegenerative conditions is to be able to edit the faulty genes causing the disease. Wood is confident that we will have the ability to do this in time. I have absolutely no doubt this approach is going to work, he says.

The current CRISPR technologies for gene editing are in their infancy and there are concerns about the accuracy of the editing process but, according to Watt, other base editing technologies that may be safer are coming down the pipeline towards readiness for clinical studies.

For now, there are still hurdles in the development of gene-silencing drugs, particularly for neurodegenerative conditions. One concern is improving the design of clinical trials and how patients are recruited, says Wood. Its really hard to take patients in whom the disease has progressed, in whom theres an inflammatory environment in the brain, and expect a positive result.

He suggests trials should concentrate on treating groups at an earlier stage who have a better chance of responding to drugs. Also important is better markers to measure disease progression and trial success.

What is it you actually measure in the patients that tells you that drug is working for something like Alzheimers?

What is it you actually measure in the patients that tells you that drug is working for something like Alzheimers? Thats quite tough, says Wood, whose team is trying to develop more sensitive cognitive and memory tests, so that it will be simpler to assess the next generation of gene-silencing drugs on their way.

Of course, it is important to remember that even if the gene-silencing drugs are able to reach targets deep in the brain, if the target is wrong, we will still fail, says Watts.

While Huntingtons disease can be linked to a single gene, the situation is less simple for Alzheimers disease. A number of recent failures of drugs that target amyloid plaques, including crenezumab in June 2022, developed by Genentech and Swiss biotech company AC Immune, have led to questions about this approach. As delivery technology improves, gene-silencing drugs may ultimately help provide an answer.

It becomes easier to cleanly address the questions of disease hypothesis, [and discover] what [we] can silence that will help a patient, says Watts.

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Silencing the genes that harm the brain - The Pharmaceutical Journal

The following discussion and analysis of our financial condition and results ofoperations should be read in conjunction with our unaudited condensedconsolidated financial statements and related notes included in this QuarterlyReport on Form 10-Q and the audited financial statements and notes thereto as ofand for the year ended December 31, 2021 and the related Management's Discussionand Analysis of Financial Condition and Results of Operations, included in ourAnnual Report on Form 10-K for the year ended December 31, 2021, or AnnualReport, filed with the Securities and Exchange Commission, or the SEC, on March31, 2022. Unless the context requires otherwise, references in this QuarterlyReport on Form 10-Q to "we," "us," and "our" refer to Taysha Gene Therapies,Inc. together with its consolidated subsidiaries.

Forward-Looking Statements

The information in this discussion contains forward-looking statements andinformation within the meaning of Section 27A of the Securities Act of 1933, asamended, or the Securities Act, and Section 21E of the Securities Exchange Actof 1934, as amended, or the Exchange Act, which are subject to the "safe harbor"created by those sections. These forward-looking statements include, but are notlimited to, statements concerning our strategy, future operations, futurefinancial position, future revenues, projected costs, prospects and plans andobjectives of management. The words "anticipates," "believes," "estimates,""expects," "intends," "may," "plans," "projects," "will," "would" and similarexpressions are intended to identify forward-looking statements, although notall forward-looking statements contain these identifying words. We may notactually achieve the plans, intentions, or expectations disclosed in ourforward-looking statements and you should not place undue reliance on ourforward-looking statements. Actual results or events could differ materiallyfrom the plans, intentions and expectations disclosed in the forward-lookingstatements that we make. These forward-looking statements involve risks anduncertainties that could cause our actual results to differ materially fromthose in the forward-looking statements, including, without limitation, therisks set forth in Part II, Item 1A, "Risk Factors" in this Quarterly Report onForm 10-Q and Part II, Item 1A, "Risk Factors" in our Annual Report. Theforward-looking statements are applicable only as of the date on which they aremade, and we do not assume any obligation to update any forward-lookingstatements.

Note Regarding Trademarks

All brand names or trademarks appearing in this report are the property of theirrespective holders. Unless the context requires otherwise, references in thisreport to the "Company," "we," "us," and "our" refer to Taysha Gene Therapies,Inc.

Overview

We are a patient-centric gene therapy company focused on developing andcommercializing AAV-based gene therapies for the treatment of monogenic diseasesof the central nervous system, or CNS, in both rare and large patientpopulations. We were founded in partnership with The University of TexasSouthwestern Medical Center, or UT Southwestern, to develop and commercializetransformative gene therapy treatments. Together with UT Southwestern, we areadvancing a deep and sustainable product portfolio of gene therapy productcandidates, with exclusive options to acquire several additional developmentprograms at no cost. By combining our management team's proven experience ingene therapy drug development and commercialization with UT Southwestern'sworld-class gene therapy research capabilities, we believe we have created apowerful engine to develop transformative therapies to dramatically improvepatients' lives. In March 2022, we announced strategic pipeline prioritizationinitiatives focused on GAN and Rett syndrome. We will conduct smallproof-of-concept studies in CLN1 disease and SLC13A5 deficiency. Development ofthe CLN7 program will continue in collaboration with existing partners withfuture clinical development to focus on the first-generation construct.Substantially all other research and development activities have been paused toincrease operational efficiency.

In April 2021, we acquired exclusive worldwide rights to TSHA-120, aclinical-stage, intrathecally dosed AAV9 gene therapy program for the treatmentof giant axonal neuropathy, or GAN. A Phase 1/2 clinical trial of TSHA-120 isbeing conducted by the National Institutes of Health, or NIH, under an acceptedinvestigational new drug application, or IND. We reported clinical safety andfunctional MFM32 data from this trial for the highest dose cohort of 3.5E14total vg in January 2022, where we saw continued clinically meaningful slowingof disease progression similar to that achieved with the lower dose cohorts,which we considered confirmatory of disease modification. We recently completeda commercially representative GMP batch of TSHA-120 which demonstrated that thepivotal lots from the commercial grade material were generally analyticallycomparable to the original clinical trial material. Release testing for thisbatch is currently underway and expected to be completed in September 2022.Additional discussions with Health Authorities are planned to discuss thesecomparability data and a potential registration pathway with feedbackanticipated by the end of 2022. For Rett syndrome, we submitted a Clinical TrialApplication, or CTA, filing to Health Canada in November 2021 and announcedinitiation of clinical development of TSHA-102 under the approved CTA in March2022. We expect to report preliminary clinical data for TSHA-102 in Rettsyndrome by year-end 2022. We recently executed an exclusive option from UTSouthwestern to license worldwide rights to a clinical-stage CLN7 program. TheCLN7 program is currently in a Phase 1 clinical

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proof-of-concept trial run by UT Southwestern, and we reported preliminaryclinical safety data for the first patient in history to be intrathecally dosedat 1.0x1015 total vg with the first-generation construct in December 2021.Development of the CLN7 program will continue in collaboration with existingpartners with future clinical development to focus on the first-generationconstruct. We will conduct small proof-of-concept studies in CLN1 disease andSLC13A5 deficiency that we believe can further validate our platform.

We have a limited operating history. Since our inception, our operations havefocused on organizing and staffing our company, business planning, raisingcapital and entering into collaboration agreements for conducting preclinicalresearch and development activities for our product candidates. All of our leadproduct candidates are still in the clinical or preclinical developmentstage. We do not have any product candidates approved for sale and have notgenerated any revenue from product sales. We have funded our operationsprimarily through the sale of equity, raising an aggregate of $319.0 million ofgross proceeds from our initial public offering and private placements of ourconvertible preferred stock as well as sales of common stock pursuant to ourSales Agreement (as defined below). In addition, we drew down $30.0 million and$10.0 million in term loans on August 12, 2021 and December 29, 2021,respectively.

On August 12, 2021, or the Closing Date, we entered into a Loan and SecurityAgreement, or the Term Loan Agreement, with the lenders party thereto from timeto time, or the Lenders and Silicon Valley Bank, as administrative agent andcollateral agent for the Lenders, or the Agent. The Term Loan Agreement providesfor (i) on the Closing Date, $40.0 million aggregate principal amount of termloans available through December 31, 2021, (ii) from January 1, 2022 untilSeptember 30, 2022, an additional $20.0 million term loan facility available atthe Company's option upon having three distinct and active clinical stageprograms, determined at the discretion of the Agent, at the time of draw, (iii)from October 1, 2022 until March 31, 2023, an additional $20.0 million term loanfacility available at our option upon having three distinct and active clinicalstage programs, determined at the discretion of the Agent, at the time of drawand (iv) from April 1, 2023 until December 31, 2023, an additional $20.0 millionterm loan facility available upon approval by the Agent and the Lenders, or,collectively, the Term Loans. We drew $30.0 million in term loans on the ClosingDate and drew an additional $10.0 million term loan on December 29, 2021. Theloan repayment schedule provides for interest only payments until August 31,2024, followed by consecutive monthly payments of principal and interest. Allunpaid principal and accrued and unpaid interest with respect to each term loanis due and payable in full on August 1, 2026.

Since our inception, we have incurred significant operating losses. Our netlosses were $84.0 million for the six months ended June 30, 2022 and $73.0million for the six months ended June 30, 2021. As of June 30, 2022, we had anaccumulated deficit of $319.6 million. We expect to continue to incursignificant expenses and operating losses for the foreseeable future. Weanticipate that our expenses will increase significantly in connection with ourongoing activities, as we:

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Our Pipeline

We are advancing a deep and sustainable product portfolio of gene therapyproduct candidates for monogenic diseases of the CNS in both rare and largepatient populations, with exclusive options to acquire several additionaldevelopment programs at no cost. Our portfolio of gene therapy candidatestargets broad neurological indications across three distinct therapeuticcategories: neurodegenerative diseases, neurodevelopmental disorders and geneticepilepsies. Our current pipeline, including the stage of development of each ofour product candidates, is represented in the table below:

TSHA-120 for Giant Axonal Neuropathy (GAN)

In March 2021, we acquired the exclusive worldwide rights to a clinical-stage,intrathecally dosed AAV9 gene therapy program, now known as TSHA-120, for thetreatment of giant axonal neuropathy, or GAN, pursuant to a license agreementwith Hannah's Hope Fund for Giant Axonal Neuropathy, Inc., or HHF. Under theterms of the agreement, HHF received an upfront payment of $5.5 million and willbe eligible to receive clinical, regulatory and commercial milestones totalingup to $19.3 million, as well as a low, single-digit royalty on net sales uponcommercialization of TSHA-120.

GAN is a rare autosomal recessive disease of the central and peripheral nervoussystems caused by loss-of-function gigaxonin gene mutations. There are anestimated 5,000 affected GAN patients in addressable markets.

Symptoms and features of children with GAN usually develop around the age offive years and include an abnormal, wide based, unsteady gait, weakness and somesensory loss. There is often associated dull, tightly curled, coarse hair, giantaxons seen on a nerve biopsy, and spinal cord atrophy and white matterabnormality seen on MRI. Symptoms progress and as the children grow older theydevelop progressive scoliosis and contractures, their weakness progresses to thepoint where they will need a wheelchair for mobility, respiratory musclestrength diminishes to the point where the child will need a ventilator (usuallyin the early to mid-teens) and the children often die during their late teens orearly twenties, typically due to respiratory failure. There is an early- andlate-onset phenotype associated with the disease, with shared physiology. Thelate-onset phenotype is often categorized as Charcot-Marie-Tooth Type 2, orCMT2, with a lack of tightly curled hair and CNS symptoms with relatively slowprogression of disease. This phenotype represents up to 6% of all CMT2diagnosis. In the late-onset population, patients have poor quality of life butthe disease is not life-limiting. In early-onset disease, symptomatic treatmentsattempt to maximize physical development and minimize the rate of deterioration.Currently, there are no approved disease-modifying treatments available.

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TSHA-120 is an AAV9 self-complementary viral vector encoding the full lengthhuman gigaxonin protein. The construct was invented by Dr. Steven Gray and isthe first AAV9 gene therapy candidate to deliver a functional copy of the GANgene under the control of a JeT promoter that drives ubiquitous expression.

We have received orphan drug designation and rare pediatric disease designationfrom the U.S. Food and Drug Administration, or the FDA, for TSHA-120 for thetreatment of GAN. In April 2022, we received orphan drug designation from theEuropean Commission for TSHA-120 for the treatment of GAN.

There is an ongoing longitudinal prospective natural history study being led bythe NIH, that has already identified and followed a number of patients with GANfor over five years with disease progression characterized by a number ofclinical assessments. The GAN natural history study was initiated in 2013 andincluded 45 patients with GAN, aged 3 to 21 years. Imaging data from this studyhave demonstrated that there are distinctive increased T2 signal abnormalitieswithin the cerebellar white matter surrounding the dentate nucleus of thecerebellum, which represent one of the earliest brain imaging findings inindividuals with GAN. These findings precede the more widespread periventricularand deep white matter signal abnormalities associated with advanced disease. Inaddition, cortical and spinal cord atrophy appeared to correspond to moreadvanced disease severity and older age. Impaired pulmonary function in patientswith GAN also was observed, with forced vital capacity correlating well withseveral functional outcomes such as the MFM32, a validated 32-item scale formotor function measurement developed for neuromuscular diseases. Nocturnalhypoventilation and sleep apnea progressed over time, with sleep apnea worseningas ambulatory function

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deteriorated. Total MFM32 score also correlated with ambulatory status, whereindependently ambulant individuals performed better and had higher MFM32 scoresthan the non-ambulant group, as shown in the graph below.

Patients also reported significant autonomic dysfunction based on the COMPASS 31self-assessment questionnaire. In addition, nerve conduction functiondemonstrated progressive sensorimotor polyneuropathy with age. As would beexpected for a neurodegenerative disease, younger patients have higher baselineMFM32 scores. However, the rate of decline in the MFM32 scores demonstratedconsistency across patients of all ages, with most demonstrating an average8-point decline per year regardless of age and/or baseline MFM32 score, as shownin the natural history plot below.

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A 4-point score change in the MFM32 is considered clinically meaningful,suggesting that patients with GAN lose significant function annually. To date,we have up to eight years of robust data from this study.

Preclinical Data

TSHA-120 performed well across in vitro and in vivo studies, and demonstratedimproved motor function and nerve pathology, and long-term safety across severalanimal models. Of note, improved dorsal root ganglia, or DRG, pathology wasdemonstrated in TSHA-120-treated GAN knockout mice. These preclinical resultshave been published in a number of peer-reviewed journals.

Additional preclinical data from a GAN knockout rodent model that had receivedAAV9-mediated GAN gene therapy demonstrated that GAN rodents treated at 16months performed significantly better than 18-month old untreated GAN rodentsand equivalently to controls. These rodents were evaluated using a rotarodperformance test which is designed to evaluate endurance, balance, grip strengthand motor coordination in rodents. The time to fall off the rotarod, known aslatency, was also evaluated and the data below demonstrated the clear differencein latency in treated versus untreated GAN rodents.

A result is considered statistically significant when the probability of theresult occurring by random chance, rather than from the efficacy of thetreatment, is sufficiently low. The conventional method for determining thestatistical significance of a result is known as the "p-value," which representsthe probability that random chance caused the result (e.g., a p-value = 0.01means that there is a 1% probability that the difference between the controlgroup and the treatment group is purely due to random chance). Generally, ap-value less than 0.05 is considered statistically significant.

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With respect to DRG inflammation, a topic of considerable interest within thegene therapy arena, the DRG have a significantly abnormal histologicalappearance and function as a consequence of underlying disease pathophysiology.Treatment with TSHA-120 resulted in considerable improvements in thepathological appearance of the DRG in the GAN knockout mice. Shown below istissue from a GAN knockout mouse model with numerous abnormal neuronalinclusions containing aggregates of damaged neurofilament in the DRG asindicated by the yellow arrows. On image C, tissue from the GAN knockout micetreated with an intrathecal (IT) injection of TSHA-120 had a notable improvementin the reduction of these neuronal inclusions in the DRG.

When a quantitative approach to reduce inclusions in the DRG was applied, it wasobserved that TSHA-120 treated mice experienced a statistically significantreduction in the average number of neuronal inclusions versus the GAN knockoutmice that received vehicle as illustrated below.

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Additionally, TSHA-120 demonstrated improved pathology of the sciatic nerve inthe GAN knockout mice as shown below.

Results of Ongoing Phase 1/2 Clinical Trial

A Phase 1/2 clinical trial of TSHA-120 is being conducted by the NIH under anaccepted IND. The ongoing trial is a single-site, open-label, non-randomizeddose-escalation trial, in which patients are intrathecally dosed with one of 4dose levels of TSHA-120 - 3.5E13 total vg, 1.2E14 total vg, 1.8E14 total vg or3.5E14 total vg. The primary endpoint is to assess safety, with secondaryendpoints measuring efficacy using pathologic, physiologic, functional, andclinical markers. To date, 14 patients have been intrathecally dosed and twelvepatients have at least three years' worth of long-term follow up data.

At 1-year post-gene transfer, a clinically meaningful and statisticallysignificant slowing or halting of disease progression was seen with TSHA-120 atthe highest dose of 3.5E14 total vg (n=3). The change in the rate of decline inthe MFM32 score improved by 5 points in the 3.5E14 total vg cohort compared toan 8-point decline in natural history.

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Although the change in the MFM32 score was clinically meaningful, we might haveexpected a greater change in the MFM32 score compared to natural history in thefirst year but one patient in the high dose cohort was a delayed responder. Atthe 12-month follow-up visit, the patient had a 7-point decline in the MFM32total score that was similar to the slope of the natural history curve as shownbelow. Notably, from Year 1 post gene transfer to Year 2, this patient's changein the MFM32 score remained unchanged suggesting stabilization of disease at 2years post-treatment. At that 2-year post treatment timepoint, there was a9-point improvement in the patient's MFM32 score compared to the estimatednatural history decline of 16 points. The annualized estimate of natural historyover time assumes the same rate of decline as in Year 1.

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An additional analysis was performed to examine the change in the rate ofdecline in the MFM32 score of all therapeutic doses combined (n=12). As shownbelow, the change in the rate of decline in the MFM32 score improved by 7 pointsby Year 1 compared to the natural history decline in the MFM32 score of 8points. This result was clinically meaningful and statistically significant.

A Bayesian analysis was conducted on the 1.2E14 total vg, 1.8E14 total vg and3.5E14 total vg dose cohorts at Year 1 to assess the probability of clinicallymeaningful slowing of disease progression as compared to natural history. Thistype of statistical analysis enables direct probability statements to be madeand is both useful and accepted by regulatory agencies in interventional studiesof rare diseases and small patient populations. As shown in the table below, forall therapeutic dose cohorts, there was nearly 100% probability of any slowingof disease and a 96.7% probability of clinically meaningful slowing of 50% ormore following treatment with TSHA-120 compared to natural history data.

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There remained consistent improvement in TSHA-120's effect over time on the meanchange from baseline in the MFM32 score for all patients in the therapeutic dosecohorts compared to the estimated natural history decline over the years. ByYear 3, as depicted below, there was a 10-point improvement in the mean changefrom baseline in MFM32 score for all patients in the therapeutic dose cohorts.

In addition to the compelling three-year data, there was one patient at Year 5whose MFM32 change from baseline improved by nearly 26-points in the 1.2E14total vg dose cohort compared to the estimated natural history decline of 40points by this timepoint.

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Below is an additional analysis of the mean change from baseline in MFM32 scorefor the therapeutic dose cohorts compared to natural history at patients' lastvisit. As shown, TSHA-120 demonstrated increasing improvement in the mean changein MFM32 score from baseline over time.

Sensory nerve action potential, or SNAP, was assessed through nerve conductionstudies in patients with GAN. Natural history data from the NIH suggest rapidand irreversible decline in sensory function early in life in patients with GAN.SNAPs are within normal limits early in life and rapid reduction in SNAPamplitude occurs around the age of symptom presentation. As demonstrated below,all patients with classic GAN have an abnormally low SNAP by the age of 4,reflective of compromised sensory neuronal function. By age 9, all patients hadan irreversibly absent SNAP. The results from these nerve conduction studiesreflect the clinical progression of patients with GAN.

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TSHA-120-treated patients demonstrated a durable improvement in SNAP responsecompared to natural history. Five of the twelve patients treated demonstrated aresponse. One patient demonstrated near complete recoverability to normal fromzero at the time of treatment.

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Once SNAP reaches zero, natural history suggests sensory function is presumednon-recoverable. Among patients treated with 1.2E14 total vg or greater ofTSHA-120, the three patients with a positive value at baseline maintained apositive SNAP at last study visit with the longest span of 3 years to date andcontinue to improve.

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Below are individual patient SNAP change from baseline from treated patients whoshowed a positive response including their run-in natural history.

Biopsies of TSHA-120-treated patients confirmed presence of regenerative nerveclusters. Below is pathology data from biopsies of the superficial radialsensory nerve in 11 out of 11 patient samples analyzed. The remaining twosamples were unable to be assessed due to biopsy limitations. Peripheral nervebiopsies from the superficial radial sensory nerve were obtained at baseline andat 1 year post gene therapy transfer. Data consistently generated an increase inthe number of regenerative clusters observed at Year 1 compared to baseline,indicating active regeneration of nerve fibers following treatment withTSHA-120. Data also indicated improvement in disease pathology, providingevidence that the peripheral nervous system can respond to treatment.

Loss of vision has been frequently cited by patients and caregivers as a symptomthey find particularly debilitating and would like to see improvement infollowing treatment. Patients were analyzed for visual acuity using a standardLogarithm of the Minimum Angle of Resolution, or LogMAR. An increase in LogMARscore represents a decrease in visual acuity. A LogMAR score of 0 means normalvision, approximately 0.3 reflects the need for eyeglasses, and a score value of1.0 reflects blindness. Based on natural history,

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individuals with GAN experience a progressive loss in visual function asindicated by an increase in the LogMAR score. Ophthalmologic assessmentsfollowing treatment with TSHA-120 demonstrated preservation of visual acuityover time compared to the loss of visual acuity observed in natural history.Stabilization of visual acuity was observed following treatment with TSHA-120 asdemonstrated below.

The thickness of the retinal nerve fiber layer or RNFL was also examined as anobjective biomarker of visual system involvement and overall nervous systemdegeneration in GAN. Treatment with TSHA-120 resulted in stabilization of RNFLthickness and prevention of axonal nerve degeneration compared to diffusethinning of RNFL observed in natural history as measured by optical coherencetomography, or OCT. Analysis by individual dose groups, as seen on the graphbelow, demonstrated relatively stable RNFL thickness which is in contrast to thenatural history of GAN, where RNFL decreases. Overall, these data provide newevidence of TSHA-120's ability to generate nerve fibers and preserve visualacuity.

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As of January 2022, there were 53 patient-years of clinical data to supportTSHA-120's favorable safety and tolerability profile. TSHA-120 has beenwell-tolerated at multiple doses with no signs of significant acute or subacuteinflammation, no sudden sensory changes and no drug-related or persistingtransaminitis. Adverse events related to immunosuppression or study procedureswere similar to what has been seen with other gene therapies and transient innature. There was no increase in incidence of adverse events with increaseddose. Importantly, TSHA-120 was safely dosed in the presence of neutralizingantibodies as a result of the combination of route of administration, dosing andimmunosuppression regimen.

We currently have up to six years of longitudinal data in individual patientswith GAN and collectively 53-patient years of clinical safety and efficacy datafrom our ongoing clinical study. Treatment with TSHA-120 was well-tolerated withno significant safety issues. There was no increase in incidence of adverseevents with increased dose, no dose-limiting toxicity, no signs of acute orsubacute inflammation, no sudden sensory changes and no drug-related orpersistent elevation of transaminases. Adverse events related toimmunosuppression or study procedures were similar to what was seen with othergene therapies and transient in nature.

We believe the comprehensive set of evidence generated across diseasemanifestations, depicted in the table below, support a robust clinical packagefor TSHA-120 in GAN.

In order to deliver a robust chemistry, manufacturing, and controls, or CMC,data package to support licensure discussions, we have successfully completedsix development and GMP lots of TSHA-120 with our contract development andmanufacturing organization, or CDMO, partner. We have also completed acomprehensive side-by-side biochemical and biophysical analysis of

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current and previous clinical lots. Our CDMO utilizes the same Pro10TMmanufacturing platform used to produce the original GAN lots, therefore reducingwhich is intended to reduce comparability risk. Five development lots rangingfrom 2L to 250L scale and one full-scale 500L GMP lot were analyzed side-by-sidewith the current TSHA-120 clinical lot using a comprehensive analytical panelthat meets current regulatory requirements including assays for criticalattributes such as product and process residuals, empty/full ratio, geneticintegrity, potency and strength.

The side-by-side analysis demonstrated that the newly produced TSHA-120 lotswere generally comparable to the original clinical trial material in impurityprofile including host cell contaminants, residual plasmid, empty particlecontent, aggregate content and genomic integrity. These results supported ourbiophysical and biochemical comparability of the newly produced lots.Furthermore, we developed product-specific GAN potency methods which have alsodemonstrated that the previous and current clinical lots were functionallyindistinguishable. Validation of our potency release assay is now underway.

We have applied our panel of release assays for side-by-side testing of theoriginal clinical trial material and our commercial grade lots. Shown below areeight of the most critical attributes of TSHA-120.

First, all results demonstrated that both the clinical and commercial grade lotswere of a high purity and lacked significant levels of host cell or processcontaminants such as protein and, DNA or and aggregated species. Vector puritywas in excess of 95% for all three lots and host cell protein contamination wasbelow detection. In addition, and aggregation of all lots was very low. Hostcell and plasmid DNA contamination are also important attributes to discuss withregulatory agencies since carryover represents a theoretical immunogenicity oroncogenicity risk. Residual plasmid and host cell DNA were similar for all lots,indicating a similar safety profile for both products. Empty capsids are a keyattribute for AAV vectors since empty capsids can stimulate immune responses tothe vector and reduce potency. All three lots were highly enriched in fullparticles. Potency of AAV vectors is a key measure that correlates with clinicalefficacy. We developed a number of product-specific potency assays to measurethe functional activity of our product which is reported relative to a referencestandard. These assays recapitulated the biological activity of TSHA-120starting with transduction of GAN knockout cell lines. Activity is measured byquantitation of transgene RNA or protein expression as two independent andcomplimentary readouts. We observed good agreement with both readouts and highactivity of all three lots against our reference suggesting that the lots are ofhigh and comparable activity.

Overall, these results support that our early clinical and pivotal lots arebiochemically and biophysically similar and based on these results we believethey should perform identically in a clinical study.

Recently, regulators have encouraged sponsors to conduct deeper analysis ofproduct contaminants not covered by standard release assays to better assessproduct safety and comparability. To comply with this guidance, we have addedPac-Bio next generation sequencing to our product characterization panel tobetter understand the nature of nucleic acid contaminants in our products. Thismethod not only allows us to identify the source of the nucleic acid, but alsothe fragment size, and sequence variability, which also needs to be consideredwhen assessing AAV safety and efficacy. Our analysis of the clinical trial lotand commercial grade pivotal batches demonstrated that the source andcomposition of transgene and contaminating host and plasmid

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DNA are nearly identical and provided further support that for a conclusion thatthe nature of our product is unchanged between our early clinical and pivotalbatches as noted in the below pie charts.

The TSHA-120 pivotal lot, which yielded over 50 patient doses of TSHA-120 at thehighest dose cohort of 3.5E14 total vg, is expected to complete quality releasetesting by end of the third quarter of 2022. This material positions us forfuture BLA-enabling activities and commercial production. These lots were alsoplaced on stability to provide critical shelf-life data in support of our BLAfiling.

In September 2021, we submitted a request for a Scientific Advice meeting forTSHA-120 to the United Kingdom's Medicines and Healthcare products RegulatoryAgency, or MHRA, and were granted a meeting in January 2022. MHRA agreed on ourcommercial manufacturing and release assay testing strategy including potencyassays and we plan to dose a few additional patients with commercial gradematerial, which will be released in September 2022. Finally, MHRA was supportiveof our proposal to perform validation work on MFM32 for GAN as a key clinicalendpoint and for us to explore the MFM32 items with patients and families aspart of this process. Given the positive comparability data for TSHA-120 that werecently received, additional discussions with Health Authorities to discussthese data and potential registration pathway are planned with regulatoryfeedback anticipated by year-end 2022.

TSHA-102 for Rett Syndrome

TSHA-102, a neurodevelopmental disorder product candidate, is being developedfor the treatment of Rett syndrome, one of the most common genetic causes ofsevere intellectual disability, characterized by rapid developmental regressionand in many cases caused by heterozygous loss of function mutations in MECP2, agene essential for neuronal and synaptic function in the brain. The estimatedprevalence of Rett syndrome is 350,000 patients worldwide and the disease occursin 1 of every 10,000 female births worldwide. We designed TSHA-102 to preventgene overexpression-related toxicity by inserting microRNA, or miRNA, targetbinding sites into the 3' untranslated region of viral genomes. Thisoverexpression of MECP2 is seen in the clinic in patients with a condition knownas MECP2 duplication syndrome, where elevated levels of MECP2 result in aclinical phenotype similar to Rett syndrome both in terms of symptoms andseverity. TSHA-102 is constructed from a neuronal specific promoter, MeP426,coupled with the miniMECP2 transgene, a truncated version of MECP2, andmiRNA-Responsive Auto-Regulatory Element, or miRARE, our novel miRNA targetpanel, packaged in self-complementary AAV9. Currently, there are no approvedtherapies for the treatment of Rett syndrome, which affects more than 350,000patients worldwide, according to the Rett Syndrome Research Trust.

In May 2021, preclinical data from the ongoing natural history study forTSHA-102 were published online in Brain, a highly esteemed neurological sciencepeer-reviewed journal. The preclinical study was conducted by the UTSouthwestern Medical Center laboratory of Sarah Sinnett, Ph.D., and evaluatedthe safety and efficacy of regulated miniMECP2 gene transfer, TSHA-102(AAV9/miniMECP2-miRARE), via IT administration in adolescent mice between fourand five weeks of age. TSHA-102 was compared to unregulated full length MECP2(AAV9/MECP2) and unregulated miniMECP2 (AAV9/miniMECP2).

TSHA-102 extended knockout survival by 56% via IT delivery. In contrast, theunregulated miniMECP2 gene transfer failed to significantly extend knockoutsurvival at either dose tested. Additionally, the unregulated full-length MECP2construct did not demonstrate a significant extension in survival and wasassociated with an unacceptable toxicity profile in wild type mice.

In addition to survival, behavioral side effects were explored. Mice weresubjected to phenotypic scoring and a battery of tests including gait, hindlimbclasping, tremor and others to comprise an aggregate behavioral score. miRAREattenuated

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miniMECP2-mediated aggravation in wild type aggregate phenotype severity scores.Mice were scored on an aggregate severity scale using an established protocol.AAV9/MECP2- and AAV9/miniMECP2-treated wild type mice had a significantly highermean (worse) aggregate behavioral severity score versus that observed forsaline-treated mice (p <0.05; at 6-30 and 7-27 weeks of age, respectively).TSHA-102-treated wild type mice had a significantly lower (better) meanaggregate severity score versus those of AAV9/MECP2- and AAV9/miniMECP2-treatedmice at most timepoints from 11-19 and 9-20 weeks of age, respectively. Nosignificant difference was observed between saline- and TSHA-102-treated wildtype mice.

miRARE-mediated genotype-dependent gene regulation was demonstrated by analyzingtissue sections from wild type and knockout mice treated with AAV9 vectors givenintrathecally. When knockout mice were injected with a vector expressing themini-MECP2 transgene with and without the miRARE element, miRARE reduced overallminiMECP2 transgene expression compared to unregulated miniMECP2 in wild typemice as shown below.

TSHA-102 demonstrated regulated expression in different regions of the brain. Asshown in the graph and photos below, in the pons and midbrain, miRARE inhibitedmean MECP2 gene expression in a genotype-dependent manner as indicated bysignificantly fewer myc(+) cells observed in wild type mice compared to knockoutmice (p<0.05), thereby demonstrating that TSHA-102 achieved MECP2 expressionlevels similar to normal physiological parameters.

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In preclinical animal models, intrathecal myc-tagged TSHA-102 was not associatedwith early death and did not cause adverse behavioral side effects in wild typemice demonstrating appropriate downregulation of miniMECP2 protein expression ascompared to unregulated MECP2 gene therapy constructs. In addition, preclinicaldata demonstrated that miRARE reduced overall expression of miniMECP2 transgeneexpression and regulated genotype-dependent myc-tagged miniMECP2 expressionacross different brain regions on a cell-by-cell basis and improved the safetyof TSHA-102 without compromising efficacy in juvenile mice. Pharmacologicactivity of TSHA-102 following IT administration was assessed in the MECP2knockout mouse model of Rett syndrome across three dose levels and three agegroups (n=252). A one-time IT injection of TSHA-102 significantly increasedsurvival at all dose levels, with the mid to high doses improving survivalacross all age groups compared to vehicle-treated controls. Treatment withTSHA-102 significantly improved body weight, motor function and respiratoryassessments in MECP2 knockout mice. An additional study in neonatal mice isongoing, and preliminary data suggest normalization of survival. Finally, anIND/CTA-enabling 6-month Good Laboratory Practice, or GLP, toxicology study(n=24) examined the biodistribution, toxicological effects and mechanism ofaction of TSHA-102 when intrathecally administered to Non-Human Primates, orNHPs, across three dose levels. Biodistribution, as reflected by DNA copynumber, was observed in multiple areas of the brain, sections of spinal cord andthe DRG. Importantly, mRNA levels across multiple tissues were low, indicatingmiRARE regulation is minimizing transgene expression from the construct in thepresence of endogenous MECP2 as expected, despite the high levels of DNA thatwere delivered. No toxicity from

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transgene overexpression was observed, confirmed by functional andhistopathologic evaluations demonstrating no detrimental change inneurobehavioral assessments and no adverse tissue findings on necropsy.

In neonatal knockout Rett mice, treatment with TSHA-102 resulted in nearnormalization of survival as shown below. A single intracerebroventricular, orICV, injection of TSHA-102 at a dose of 8.8E10 vg/mouse (Human Equivalent Doseof 2.86E14 vg/participant) within 48 hours after birth in Mecp2-/Y male micesignificantly extended the survival of the animals as shown below. All cohorts,including vehicle, were sacrificed at 34 weeks. Preliminary data demonstratedapproximately 70% of the treated Mecp2-/Y males survived to 34 weeks of agecompared to 9 weeks in the vehicle-treated Mecp2-/Y male.

In addition, neonatal knockout Rett mice demonstrated normalization of behaviorfollowing treatment with TSHA-102 as assessed by the Bird Score, a compositemeasure of six different phenotypic abilities. Knockout animals were initiallyassessed at 4 weeks of age with a mean Bird Score of 4. Over the course of thestudy, TSHA-102 improved the behaviors (as assessed by the Bird aggregate score)of TSHA-102 treated mice as shown below.

In summary, we believe the totality of preclinical data generated to date,specifically including the mouse pharmacology study to ascertain minimallyeffective dose, the two toxicology studies (wild type rat and wild type NHP) andthe recent mouse neonatal data, represents the most robust package supportingclinical advancement of TSHA-102 in Rett syndrome as shown below.

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Safety and biodistribution assessments in NHPs were presented in May 2022 at theInternational Rett Syndrome Foundation (IRSF) meeting along with the caregiverperspective on Rett syndrome in adulthood. At the ASCEND National Summit, therewas an oral presentation on "Putting Patients at the Center." Finally, mousepharmacology, rat and NHP toxicology data were presented at the 25th AnnualMeeting of the American Society of Gene & Cell Therapy (ASGCT).

We submitted a CTA for TSHA-102 in November 2021 and announced initiation ofclinical development under a CTA approved by Health Canada in March 2022. We areadvancing TSHA-102 in the REVEAL Phase 1/2 clinical trial which is anopen-label, dose escalation, randomized, multicenter study that will examine thesafety and efficacy of TSHA-102 in adult female patients with Rett syndrome. Upto 18 patients will be enrolled. In the first cohort, a single 5E14 total vgdose of TSHA-102 will be given intrathecally. The second cohort will be given a1E15 total vg dose of TSHA-102. Key assessments will include Rett-specific andglobal assessments, quality of life, biomarkers, and neurophysiology and imagingassessments. Sainte-Justine Mother and Child University Hospital Center inMontreal, Quebec, Canada has been selected as the initial clinical trial siteunder the direction of Dr. Elsa Rossignol, Assistant Professor Neuroscience andPediatrics, and Principal Investigator. We expect to report preliminary clinicaldata for TSHA-102 in Rett syndrome by year-end 2022.

We have received orphan drug designation and rare pediatric disease designationfrom the FDA and orphan drug designation from the European Commission forTSHA-102 for the treatment of Rett syndrome.

TSHA-121 for CLN7 Disease

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