Search Results for: tooth regeneration fda approval

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

--------------------------------------------------------------------------------

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:

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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,

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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

--------------------------------------------------------------------------------

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

--------------------------------------------------------------------------------

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

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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

--------------------------------------------------------------------------------

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.

--------------------------------------------------------------------------------

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

More:
TAYSHA GENE THERAPIES, INC. Management's Discussion and Analysis of Financial Condition and Results of Operations. (form 10-Q) - Marketscreener.com

Abstract

Gene therapy is defined as the treatment of disease by transfer of genetic material into cells. This review will explore methods available for gene transfer as well as current and potential applications for craniofacial regeneration, with emphasis on future development and design. Though non-viral gene delivery methods are limited by low gene transfer efficiency, they benefit from relative safety, low immunogenicity, ease of manufacture, and lack of DNA insert size limitation. In contrast, viral vectors are natures gene delivery machines that can be optimized to allow for tissue-specific targeting, site-specific chromosomal integration, and efficient long-term infection of dividing and non-dividing cells. In contrast to traditional replacement gene therapy, craniofacial regeneration seeks to use genetic vectors as supplemental building blocks for tissue growth and repair. Synergistic combination of viral gene therapy with craniofacial tissue engineering will significantly enhance our ability to repair and replace tissues in vivo.

Keywords: gene therapy, gene transfer, vector design, tissue engineering, virus, regeneration

Human gene therapy is defined as the treatment of disorder or disease through transfer of engineered genetic material into human cells, often by viral transduction. Since the introduction of science fiction, the popular press has toyed with the notion of viral gene delivery and its terrifying implications. One of the more recent popular works on the topic is the 2007 remake of Richard Mathesons classic 1954 novel I Am Legend, which details events following the discovery, release, and mutation of a genetically re-engineered measles virus that was initially hailed as the cure for cancer (Matheson, 1954; Lawrence, 2007). This adapted novel, which has been redone in three instances as a feature film, outlines the seemingly inevitable worldwide destruction that could result from viral gene therapy. With an emotionally stirring history of fictional violence and a debate that provokes both moral and medical issues, it may be surprising that, since 1990, billions of dollars have been spent on hundreds of human viral gene therapy clinical trials. Our society is in the midst of a paradigm shift that began with the discovery of viruses as dangerous infectious agents and will end with the use of viruses to cure disease and regenerate tissues.

On January 19, 1989, the director of the National Institutes of Health (NIH), Dr. James A. Wyngaarden, approved the first clinical protocol to insert a foreign gene into the immune cells of persons with cancer (Roberts, 1989). On September 14, 1990, W. French Anderson and his colleagues at the NIH performed the first approved gene therapy procedure on a four-year-old girl born with severe combined immunodeficiency (SCID) (Anderson, 1990). Despite the viral horror stories written by the popular media, this initial trial was largely a success, and the most recent report on this individual in 2004 noted that she is thriving as an 18-year-old teenager in suburban Cleveland (Springen, 2004). Over the next ten years, 300 clinical gene therapy trials were performed on about 3000 individuals (McKie, 2000). The field was then blackened with the death of an 18-year-old male four days after the introduction of 38 trillion particles of recombinant adenovirus into his liver (Somia and Verma, 2000). Despite this tragedy, we continue to move forward because of the great promise of novel genetic treatments that, when perfected, will likely outshine current methods, such as protein therapy or pharmacotherapeutics, for treatment of many diseases and defects.

We are now nearing the 20-year mark since the first gene therapy trial. Though success has been limited, the future still seems overwhelmingly promising, and we are steadily approaching an acceptable safety record. This review will explore non-viral and viral methods available for transgene introduction as well as their current and potential applications for craniofacial regeneration and therapy, with emphasis on future development and design.

Though this review will focus mostly on viral methods of gene delivery, it is essential to recognize that many advances have been made in the field of non-viral gene therapy. Polymeric gene delivery is desired because of its relative safety, low immunogenicity and toxicity, ease of administration and manufacture, and lack of DNA insert size limitation (Park et al., 2006). The main disadvantage is insufficient gene transfer efficiency due to the need for post-uptake endosomal escape and nuclear translocation of the DNA complex (Park et al., 2006). In this respect, clinical efficiency and specificity standards have not yet been met.

The main strategy for most synthetic polymer delivery systems is to generate cationic polymers to interact electrostatically with and neutralize negatively charged DNA (Park et al., 2006). This facilitates properties such as protection from DNAses. If a net positive charge is maintained, the polymer/DNA complex can adhere to the cell surface glycocalyx and be internalized by endocytic mechanisms. Unfortunately, the use of endocytic uptake from the external environment perpetuates the need for endocytic escape into the cytosol. This challenge in the polymeric gene delivery field has been addressed by multiple strategies, including incorporation of fusogenic peptides for endosomal membrane binding and disruption (Cho et al., 2003) and by balancing a hydrophobic cholesterol group with hydrophilic polymers to enhance escape (Mahato et al., 2001).

One of the first polymers recognized for its ability to form nanoparticulate polyelectrolyte complexes with DNA was Poly L-lysine (PLL) (Laemmli, 1975). Unfortunately, this cationic material was found to have high cytotoxicity (Choi et al., 1998) and a tendency to aggregate and precipitate (Liu et al., 2001). The solution to this dilemma was found in the form of the flexible, water-soluble polymer polyethylene glycol (PEG). Covalent coupling of PEG, or PEGylation, of a target molecule such as PLL limits its cytotoxicity and non-specific protein adsorption (Choi et al., 1998). This strategy has also been used with polyethyleneimine (PEI), a cationic gene carrier with superior transfection efficiency and unique buffering properties (Boussif et al., 1995), similarly to reduce the extent of inter-particular aggregation (Mishra et al., 2004; Quick and Anseth, 2004).

In addition to improving the bio-properties of PLL and PEI, PEGylated polymers can be conjugated to specific targeting moieties, such as sugars, antibodies, peptides, and folate (Lee and Kim, 2005). For example, peptide conjugation of the apoB-100 fragment of low-density lipoprotein can increase transfection efficiency in bovine aorta and smooth-muscle cells 150- to 180-fold (Nah et al., 2002), and RGD peptides can allow for increased selection of endothelial cells (Kim et al., 2005). To summarize, synthetic PEGylated polymers such as PLL and PEI are promising gene delivery molecules. Future study in this field is focused on biodegradable polycations such as poly(-amino ester), poly(2-aminoethyl propylene phosphate), and degradable PEI to decrease cytotoxicity and increase transfection efficiency (Akinc et al., 2003).

The natural polymer family contains materials such as cyclodextrin, chitosan, collagen, gelatin, and alginate. When compared with synthetic materials, natural polymers have the advantage of innate environmental responsiveness and the ability to be degraded and remodeled by cell-secreted enzymes. They are non-toxic at both low and high concentrations, are readily incorporated into oral or bolus matrix delivery systems, and can serve as tissue engineering scaffolds (Dang and Leong, 2006). The simplicity of oral delivery and mucoadhesive properties of materials such as chitosan make it an interesting potential polymer for gene delivery and vaccination (Roy et al., 1999). The transfection efficiency of natural polymers such as cyclodextrin, though significantly less than that of virus, is similar to that of PEI and lipofectamine (Gonzalez et al., 1999). Thus, while natural polymers benefit from degradation and remodeling, they still face significant transfection issues due to the requirement for endosomal escape. However, this strategy has been successfully used to increase bone regeneration with polymer gene activated matrix containing DNA encoding parathyroid hormone (Bonadio et al., 1999; Chen et al., 2003)

Viruses have undergone millions of years of evolution and are a species-conserved way of introducing DNA to cells (Dewannieux et al., 2006). Scientists are now attempting to fine-tune these gene delivery vehicles for treatment of human disease and defects. Regardless of method selection, there are three universal requirements for viral gene therapy vectors. First, the delivery system must be safe and immunologically inert. Second, it must protect the genetic material from degradation. Third, the vector must encode an effective therapeutic gene that has sustained expression at a defined target site. For true commercial application, the packaged vector must also be easily produced and processed and have a reasonable shelf-life. As we near the 20-year mark from the first human gene therapy clinical trial, significant advances have been made in satisfying these three requirements. However, new objectivessuch as tissue-specific targeting, site-specific chromosomal integration, and controlled infection of both dividing and non-dividing cellshave emerged. Though negative publicity has attached a significant stigma to viral gene therapy, it is indeed the most efficient method of gene transfer, and basic research and clinical trials are rapidly moving to overturn the safety concerns.

Our society is in the midst of a paradigm shift that began with the discovery of viruses as dangerous infectious agents and will end with the use of viruses to cure disease and regenerate tissues. However, safety concerns still limit the universal acceptance of this strategy. These concerns include the accidental generation of replication competent viruses during vector production and the packaging or mobilization of the engineered vector by endogenous retroviruses present in the human genome (Connolly, 2002). Either of these could lead to horizontal dissemination of new viruses from gene therapy patients. Localized concerns include random insertion or mutagenesis of the vector leading to cancer, or germ cell alteration resulting in vertical inheritance of the acquired gene (Connolly, 2002). The need for controlled genome integration hit home when two of the 11 persons treated for X-SCID with retrovirally transduced stem cells developed leukemia due to insertion of the transgene near the oncogenic gene LMO2 (Kaiser, 2003). Site-specific chromosomal integration, conditional expression of the transgene only in target cells, and the use of self-inactivating (SIN) retroviral vectors have been proposed (Yu et al., 1986) and may significantly improve the safety of viral therapy. The following sections will review the use of viral vectors for in vivo therapy, emphasizing the construction and advantages of different viruses.

Before we can successfully manipulate retroviral vectors, the composition of their genome must be thoroughly understood. Since the discovery of retroviruses in 1910, when Peyton Rous induced malignancy in chickens by the injection of cell-free filtrates from muscle tumor (VanEpps, 2005), we have gained much insight into their mechanism of action. Three main classes of recombinant retroviruses are used as tools in gene delivery: -retroviruses, lentiviruses, and spumaviruses (Chang and Sadelain, 2007). Despite their negative press, exogenous retroviruses have been used in many biological studies and facilitated the discovery of proto-oncogenes (Martin, 2004), the manipulation and investigation of intracellular pathways, and successful ex vivo treatment of persons with hemophilia and SCID (Sumimoto and Kawakami, 2007; Chu et al., 2008; Scheller et al., 2008). Retroviruses are 80- to 100-nm enveloped viruses that contain linear, non-segmented, single-stranded RNA. Retroviruses are naturally self-replicating for viral assembly and re-infection (Kurian et al., 2000). Reverse transcription allows for the generation of double-stranded DNA from the transduced 7- to 12-kBp RNA and subsequent insertion into the genome. Exogenous retroviruses can be subdivided into simple and complex categories based on the composition of their RNA vector. Simple vectors contain three basic genesgag, pol, and envwhich are necessary for viral replication and must be removed prior to gene therapy (Buchschacher, 2001) (). Identical long terminal repeats (LTRs) are present at each end of the retroviral genome. The LTRs contain promoter, enhancer, and integration sequences which facilitate interaction with attachment sites via integrase (Engelman, 1999). Complex retroviruses contain up to 15 additional accessory genes, such as tat, the transcriptional transactivator for HIV-1, vif, rev, nef, etc. (Frankel and Young, 1998).

Retroviral vector development for increased efficiency and targeting. (A) Structure of a simple retroviral genome containing coding sequences for gag, pro, pol, and env for replication. (B) Structure of a simple -retroviral gene therapy vector ...

The use of retrovirus as a gene delivery system necessitates re-engineering of the viral genome to block autonomous replication while maintaining integration efficiency. This is accomplished via the maintenance of cis-acting and removal of trans-acting factors. Five essential components for successful viral gene expression by any LTR-driven -retroviral vector include dual-LTRs, att site, primer binding site, signal psi, and the polypurine tract (Zhang and Godbey, 2006) (). The polypurine tract aids in transport of the pre-integration complex to the nucleus and allows for internal initiation of second-strand DNA synthesis (Zennou et al., 2000). Most -retroviral vectors rely on their LTRs to drive robust and ubiquitous transgene expression. Replacement of these with site-specific constitutive promoters is currently limited by expression strength and promoter silencing, but remains an active area of research that could allow for customization of transgene expression level and location. Additional vector design strategies to enhance gene expression and reduce silencing include: deletion of silencing elements (Zufferey et al., 1998), incorporation of robust promoters such as U3 or PGK (Chang and Sadelain, 2007), incorporation of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance mRNA transcript stability (Loeb et al., 2002), incorporation of scaffold or matrix attachment regions for anchorage of chromatin with stabilization of chromosomal loops (Agarwal et al., 1998), and use of insulators such as the chicken -globin locus control region to limit position effect variegation (West et al., 2002) (). Expression in bacterial plasmid form can be used for easy amplification and incorporation of drug resistance or response genes to facilitate selection ex vivo or expression in vivo (Delviks and Pathak, 1999; Jaalouk et al., 2000). Addition of the gene of interest or resistance gene under the control of a tissue-specific promoter results in a dual-promoter vector designed to enhance selection and integration. However, simultaneous use of two promoters can result in significantly reduced expression of both due to promoter interference (Apperley et al., 1991). SIN vectors which develop a defective promoter in the viral LTR have been used to circumvent this effect (Buchschacher, 2001) (). Understanding and engineering of these vectors is rapidly advancing. For example, it has been shown that ex vivo transduction of hematopoietic stem cells can be improved through control of cell-cycle stage during virus delivery (Korin and Zack, 1998) and the use of proteosome inhibitors (Goff, 2004).

Retroviruses require genome integration of their vector to function, and most, excluding lentiviruses such as HIV-1, are able to infect only dividing cells (Lewis and Emerman, 1994). Stable integration of retroviral vectors is known to occur near expressed genes and appears to be non-random (Mitchell et al., 2004). This allows for long-term expression of the transgene and makes retroviruses the vector of choice for ex vivo and in vivo transduction of highly replicative populations such as tumor cells and hematopoietic cells for treatment of chronic disease and genetic deficiency (Somia and Verma, 2000). Indeed, LTR-driven -retroviral vectors have been used in over 45 ex vivo clinical trials to treat diseases such as hemophilia, SCID, and leukemia (Kohn et al., 2003; NIH, 2008b). To advance gene therapy with retroviral vectors, increased transduction efficiency and gene expression, site-specific chromosomal integration, and cell-specific targeting are necessary. Ex vivo or in vivo -retroviral transduction of rapidly dividing cells at sites of wound healing and new bone synthesis may be used in the future to enhance craniofacial tissue regeneration. Expansion of this field to include lentiviral vectors targeting non-dividing cells could allow for the long-term restoration of non-functional salivary gland tissue or repair of quiescent periodontal defects.

Adenoviridae were initially isolated by Wallace Rowe in 1953 from adenoid explants as the virus of the common cold (Rowe et al., 1957; Ginsberg, 1999). They have since become attractive tools for gene therapy, given that their infection is generally self-limiting and non-fatal (Zhang and Godbey, 2006). Adenoviruses are the largest non-enveloped virus and contain linear, double-stranded DNA. Over 50 serotypes have been identified, but the most common in nature and in adenoviral gene therapy are group C human serotypes 2 and 5 (Barnett et al., 2002). The icosahedral adenoviral capsid is made up of hexon and penton proteins, knobbed fibers, and stabilizing minor cement proteins. These surround the core proteins and large 36-kBp adenoviral genome (Verma and Somia, 1997). The ends of the genome have inverted terminal repeats which flank a coding region capable of encoding more than 30 viral genes (Zhang and Godbey, 2006). These genes are termed early or late, depending on their temporal expression. Early genes function as regulatory proteins for viral replication, while late genes encode structural proteins for new virus assembly (Zhang and Godbey, 2006). Entry of adenovirus into the cell occurs when penton base proteins bind integrins for clathrin-mediated endocytosis. Subsequent disruption of the endosome and capsid allows for viral core entry into the nucleus (Russell, 2000). Initiation of the immediate early infection phase activates transcription of the E1A gene, a trans-acting transcriptional regulatory factor that is required for early gene activation (E1B, E2A, E2B, E3, E4, viron proteins) (Russell, 2000). The final late infection phase activates genes L1 to L5 through complex splicing; viral particles which accumulate in the nucleus are then released via cell lysis (Zhang and Godbey, 2006).

During the engineering of adenoviral vectors for gene delivery, up to 30 kbp of the 36-kbp genome can be replaced with foreign DNA (Smith, 1995). Multiple strategies have been used to produce replication-defective, transforming adenoviral vectors. In first-generation adenoviral vectors, E1 and E3 genes are deleted to allow for a 6.5-kbp insertion. However, cell-line endogenous expression of E1 can lead to E2 expression and viral replication at low levels (Russell, 2000). Other first-generation vectors have used deletion of E2 and E4 regions to allow for an insert of greater than 6.5 kbp (Lusky et al., 1998). These vectors are impaired by limited expression and a robust inflammatory response (Khan et al., 2003). Second-generation gutless vectors appear to be the most promising. Gutless vectors retain only the inverted terminal repeats and packaging sequence around the transgene (Russell, 2000). This results in prolonged transgene expression, increased insert size allowance, and reduced immune response (Fleury et al., 2004). Studies have shown that adenoviral gene expression occurs via episome formation, and only 1 in 1000 infectious units can integrate into the genome (Tenenbaum et al., 2003). Though this decreases the risk of insertional mutagenesis, it also limits adenoviral application to high-level transient transgene expression, because the gene is often lost 5 to 20 days post-transduction (Dai et al., 1995). Adenoviruses have the significant advantage of being able to infect both dividing and non-dividing cells (Verma and Somia, 1997). This makes them specially suited for applications involving brain, eye, lung, pancreas, hepatocytes, neurons, and monocytes (Blomer et al., 1997; Kafri et al., 1997). PEGylation and expression of targeting ligands on the viral capsid are being investigated to decrease the immune response and enhance targeting of adenoviral vectors (Eto et al., 2008). A clinical trial with adenovirus to repair salivary gland tissue post-radiation therapy is ongoing (Baum et al., 2006; NIH, 2008b).

Adeno-associated virus (AAV) is a parvovirus of the Dependovirus genus that was discovered in 1965 as a co-infecting agent of adenovirus preparations (Carter, 2005). The first infectious clone of AAV serotype 2 to be used for human gene therapy was generated in 1982 (Samulski et al., 1982). Since that time, AAV serotypes 1-12 and over 100 AAV variants have been isolated (Wu et al., 2006). AAV is a non-enveloped DNA virus with a 22-nm icosahedral capsid containing a 4.7-kBp linear single-stranded DNA genome. Coding capacity is limited to 4.5 kBp, but may be extended by splitting the sequence between 2 viruses that can later concatamerize after transduction (Nakai et al., 2000). The genome contains 2 unique open reading frames (ORFs) which encode 4 replication proteins and 3 capsid proteins, respectively (Ding et al., 2005). Inverted GC-rich self-complementary terminal repeats flank the ORFs and are the only cis-acting factors required for genome replication and packaging (Ding et al., 2005). Two ORF-encoded trans-acting proteins required for viral replication are rep, which controls viral replication and integration, and cap, which encodes structural components of the capsid. Though site-specific integration on chromosome 19 occurs if rep is maintained (Kotin et al., 1990), it is generally removed from rAAV-engineered vectors. Advantages of AAVs include low immunogenicity, lack of pathogenicity, a wide range of infectivity with potential cell-/tissue-specific targeting, and the ability to establish long-term latent transgene expression in both dividing and non-dividing cells.

AAVs are naturally replication-deficient and require a helper virus for replication and dissemination (Zhang and Godbey, 2006). Self-limiting infection, coupled with their ability to stably infect dividing and non-dividing cells, makes them an excellent target for in vivo gene therapy and craniofacial applications. It is generally accepted that AAV vectors persist as non-integrated circular episomal concatemers, and research shows an integration frequency of less than 1 in 30 million particles in studies of AAV delivery to muscle in rabbits (Schnepp et al., 2003; Schultz and Chamberlain, 2008). Infection occurs through binding of viral proteins to charged heparin sulfate proteoglycans (Summerford and Samulski, 1998) and is potentially enhanced by interactions with alpha-V-beta-5 integrins (Summerford et al., 1999) and human fibroblast growth factor receptor-1 (Qing et al., 1999). After clathrin-mediated endocytosis, endosomal escape, and nuclear translocation, AAV can produce latent long-term infection via episome formation in which the transgene reaches maximum expression levels after an incubation period of 4 to 8 wks and remains stable for up to 2-3 yrs in animal models (Thomas et al., 2004; Manno et al., 2006). Though it is becoming less of an issue, one of the challenges facing viral engineers is the large-scale amplification of AAVs. Baculovirus expression systems for rAAV2 vector production in SF9 cells show promise for large-scale production (Urabe et al., 2002). However, current clinical trials are limited by their reliance on transient production systems and still require complete elimination of helper virus during production (Kay et al., 2000).

The primary goal for rAAV engineering is to improve transduction efficiency to decrease vector loading while increasing target specificity. An initial successful effort to improve transduction was a switch from single-strand to self-complementary recombinant AAV vectors, to bypass the rate-limiting second-strand DNA synthesis step (McCarty et al., 2001). Because of high variation among capsids, AAV vectors have inherent tissue-targeting abilities that can be enhanced with capsid re-engineering. For example, AAV6 demonstrates increased transduction efficiency in skeletal muscle (Gao et al., 2002), and AAV4 shows preference for the CNS (Davidson et al., 2000). DNA shuffling and cloning technologies are currently being used to generate extensive libraries of recombinant AAVs that display diverse tissue specificity and potential to evade host-neutralizing antibodies (Perabo et al., 2006; Li et al., 2008). The crystal structure of the AAV2 capsid was solved in 2002 (Xie et al., 2002). AAV virons have icosahedral capsids made of 60 copies of VP1, VP2, and VP3 proteins encoded by the second genomic ORF in a variable predicted ratio of 1:1:18 (Muzyczka and Warrington, 2005). VP1 and VP2 are variable between AAV serotypes (Wu et al., 2006). Mosaic vectors (capsid structure derived from subunits of different serotypes) or chimeric vectors (capsid proteins modified by domain or amino acid swapping between serotypes) have been generated through trans-capsidation or marker-rescue/domain-swapping (Wu et al., 2006) () to enable the infection of tissues refractory to transduction by naturally occurring AAV vectors or to limit AAV infection to specific tissues (Wu et al., 2006). Insertion of peptide ligands, conjugate-based targeting, and presentation of large protein ligands on the AAV capsid are additional strategies that have been used to enhance targeting and transduction of rAAVs (Muzyczka and Warrington, 2005) (). Insertion sites for peptide-encoding DNA sequences are limited to maintain infectivity of the viron. A 14-residue core RGD peptide motif insertion is possible in VP3 at residues 261, 381, 447, 459, 573, 584, 587, and 588 (Girod et al., 1999; Shi and Bartlett, 2003) (). Integrin-RGD interactions could be exploited by craniofacial tissue engineers to enhance infection of endothelial cells and localization of rAAV to matrix-laden sites such as bone and tooth.

AAV capsid engineering for enhanced transduction and tissue-specific targeting. (A) Mosaic vectors (capsid structure derived from subunits of different serotypes) or chimeric vectors (capsid proteins modified by domain or amino acid swapping between serotypes) ...

There are three main strategies for gene delivery: in vivo, in vitro, and ex vivo. Though the most direct method is in vivo injection, this approach lacks the improved patient safety of in vitro and ex vivo methods. Systemic delivery is desirable if the target tissue is not directly accessible. However, this method often results in low specificity of gene expression, risks of toxicity due to the high vector concentration required, and potential damage to the function of healthy tissues (Zhang and Godbey, 2006). Alternatively, matrix-based delivery allows for tissue-specific gene delivery, higher localized loading of DNA or virus, and increased control over the structural microenvironment (Dang and Leong, 2006). Thus far, human in vivo clinical trials have introduced adenovirus, AAV, retrovirus, and herpes simplex virus by intravenous (IV) injection, intra-tissue injection, or lung aerosol (Kemeny et al., 2006). In contrast, ex vivo trials have focused on stable retroviral transduction of rapidly dividing populations such as CD8+ T-cells, hematopoietic stem cells, hepatocytes, and fibroblasts, followed by IV or local re-introduction. At the time of this publication, a search of the NIH Genetic Modification Clinical Research Information System (GeMCRIS) revealed 908 total gene therapy clinical trial entries in the database (NIH, 2008a). At clinicaltrials.gov, a search for interventions with gene transfer OR gene therapy returned 174 studies, of which 145 are viral-based, with 84 active, 48 completed, and 7 terminated. This cross-section of results translates to 1605 persons who have participated in this subset of completed gene therapy trials and nearly 5000 total active or anticipated participants, based on each studys documented enrollment since 1990. The following sections will briefly review the progress of gene therapy since 1990.

Gene therapy is specially suited for long-term delivery of a transgene to persons with a single genetic deficiency that is not amenable to protein or pharmacokinetic therapy. This was the premise of the first successful gene therapy clinical trials that inserted genes ex vivo into CD34+ cells to treat persons with SCID (Anderson, 1990; Blaese et al., 1993). Amazingly, persistence of the adenosine deaminase (ADA) transgene was noted in peripheral blood leukocytes 12 yrs post-therapy without adverse events (Muul et al., 2003). Since 1990, clinical treatment of genetic diseasesincluding cystic fibrosis, hemophilia, Leber congenital amaurosis, muscular dystrophy, ornithine transcarbamylase deficiency, Pompe disease, and Gauchers diseasehas been attempted, with promising documented success (Aiuti et al., 2007; Alexander et al., 2007). Following the SCID trials, treatment of cystic fibrosis by re-introduction of the cystic fibrosis transmembrane regulator (CFTR) chloride ion channel to lung epithelial cells was highly targeted and was the first use of rAAV in humans (Flotte et al., 2003). However, like many other in vivo and ex vivo clinical trials, transduction efficiency was generally insufficient to improve clinical parameters significantly. Apart from SCID, the most promising documented results for genetic deficiency correction have been the replacement of factor IX (F-IX) in hemophilia. Studies by Avigen Inc. have examined rAAV2-mediated F-IX delivery to the liver. In dogs, therapeutic levels of F-IX were achieved for multiple years following vector treatment (Manno et al., 2006). In humans, delivery of rAAV2.F-IX through the hepatic artery achieved therapeutic levels of F-IX expression for approximately 8 wks (Aiuti et al., 2007). It appears that cell-mediated immunity to the rAAV2 capsid limits expression in humans. Thus, immunomodulation and capsid engineering may make F-IX therapy a near-future reality (Krebsbach et al., 2003; Manno et al., 2006). Gene therapy is also highly desired for the treatment of neurologic and other chronic disease. Clinical trials have been implemented and/or completed for the treatment of HIV/AIDS, arthritis, angina pectoris, solid tumors, Parkinsons disease, Huntingtons disease, Alzheimers disease, Batten disease, Canavan disease, and familial hypercholesterolemia (Aiuti et al., 2007; Alexander et al., 2007; NIH, 2008a,b). Despite the many hurdles, most clinical trials are progressing steadily, with treatments for angina pectoris (Henry et al., 2007), prostate cancer (Freytag et al., 2007), non-small-cell lung cancer, and head and neck cancer now entering phase III clinical trials (NIH, 2008b).

More than 85% of the United States population requires repair or replacement of a craniofacial structure, including bone, tooth, temporomandibular joint, salivary gland, and mucosa. Regeneration of oral and craniofacial tissues presents a formidable challenge that requires synthesis of basic science, clinical science, and engineering technology. Identification of appropriate scaffolds, cell sources, and spatial and temporal signals are necessary to optimize development of a single tissue, hybrid organs consisting of multiple tissues, or tissue interface. In contrast to traditional replacement gene therapy, craniofacial regeneration via gene therapy seeks to use genetic vectors as supplemental building blocks for tissue growth and repair. Synergistic combination of viral gene therapy with craniofacial tissue engineering will significantly enhance our ability to repair and regenerate tissues in vivo.

Though the treatment of HNSCC does not directly fall in the category of craniofacial regeneration, it is the most well-developed use of gene therapy in the craniofacial region. There are three main strategies to target any solid tumor with gene therapy. First, immunomodulatory therapy seeks to increase the visibility of the tumor cells to the immune system in vivo or to modify the effector cells ex vivo to increase targeting of the tumor via the introduction of specific gene expression. In 2007, the dendric cell vaccine Provenge was deemed safe and preliminarily approved by the FDA advisory panel in a 13 to 4 vote for the treatment of prostate cancer. However, it was later denied final approval and is currently being re-evaluated (Moyad, 2007). Second, oncolytic viruses have been developed that can selectively target, multiply in, and destroy cancer cells (Dambach et al., 2006). A phase II clinical trial of OncoVex (GM-CSF), with combined chemoradiotherapy in locally advanced head and neck cancers, is ongoing (Aiuti et al., 2007). In addition, the H101 oncolytic adenovirus has undergone phase I-III clinical trials for treating head and neck cancer and is now approved for use in China (Yu and Fang, 2007). Third, suicide genes such as herpes simplex thymidine kinase can be introduced to cancer cells to increase their susceptibility to anti-viral drugs such as acyclovir (Niculescu-Duvaz and Springer, 2005). As mentioned above, application of these methods for treatment of various cancers comprises the majority of the current phase III clinical gene therapy trials (NIH, 2008b). Additional strategies of interest for specific targeting of HNSCC include local viral introduction of genes encoding p53 (Clayman et al., 1999; Yoo et al., 2004), endostatin (Lin et al., 2007), and non-viral IL-2/IL-12 (OMalley et al., 2005).

Animal-model-based gene therapy and engineering of individual craniofacial structures such as bone and cartilage have firmly established a productive relationship, and novel approaches to regeneration of complex mineralized tissues such as tooth (Nakashima et al., 2006) and TMJ (Rabie et al., 2007) are just beginning to emerge. Clinical protein delivery of PDGF-B or bone morphogenetic proteins (BMPs) at periodontal defect sites is well-known to enhance repair and healing of bone and gingiva (Kaigler et al., 2006). Gene delivery can allow for localized sustained protein expression at therapeutic levels and can overcome recombinant protein delivery issues such as cost, half-life, supra-physiologic dosing, and poor retention. In support of this, studies have shown that use of adenovirus expressing PDGF-B for treatment of periodontal defects demonstrates better results than continuous protein therapy (Jin et al., 2004; Franceschi, 2005) (). Adenoviral-, retroviral-, and AAV-mediated delivery of osteogenic genes has been demonstrated to enhance fracture repair and intramembranous or endochondral bone formation in vivo in animal models (). To meet clinical needs, gene delivery must be safe, simple, and cost-effective. Thus, focus on in vivo strategies which avoid primary cell isolation and long-term culture is ideal. An expedited ex vivo bone regeneration strategy has recently been proposed in which explants of adipose tissue or muscle directly transduced with Ad.BMP-2 without culture can be re-implanted at defect sites to enhance regeneration of critical-sized rat femoral defects (Betz et al., 2008) (). In addition, studies to release virus directly from biomaterials have been effective for bone regeneration in animal models (Hu et al., 2007)

Virally Transduced Genes for Regeneration of Craniofacial Tissues

Gene therapy for bone regeneration. (A) An expedited ex vivo bone regeneration strategy has recently been proposed in which explants of adipose tissue or muscle can be directly transduced with Ad.BMP-2 without culture. This has shown ...

Inducible vector systems, use of rAAV, and transduction of novel osteogenic factors have outstanding potential for mineralized tissue regeneration. In addition to vector design and capsid engineering for cell-specific transduction, we must now consider the use of systemic drug-inducible vector components. For example, an early study using a retroviral vector demonstrated dexamethasone-inducible GFP expression from transduced BMSCs in vitro (Jaalouk et al., 2000). Researchers have gone on to explore doxycycline-inducible tetON promoter systems. In these systems, selective induction of BMP-2 or BMP-4 expression achieved by administration of oral doxycycline can allow for localized induction of bone formation only at vector-containing sites in vivo (Gafni et al., 2004; Peng et al., 2004) (). The field of rAAV-mediated bone repair is rapidly advancing and promises superior safety, tissue targeting, and high in vivo transduction efficiency of non-dividing cells. In the past 5 years, proof-of-principle studies have been completed and have shown positive results for AAV transduction of bone-forming cells and enhanced healing of osseous defects from in vivo application of rAAV expressing constitutively active activin receptor-like kinase-2 (caAlk2), VEGF/RANKL, and ex vivo BMP-7 (Kang et al., 2007; Ulrich-Vinther, 2007) (). The use of caAlk2, a receptor that mediates BMP signaling, is emerging as an interesting gene therapy target, because of its low required therapeutic expression level and inability to be blocked by native BMP antagonist noggin and chordin (Zhang et al., 2003; Koefoed et al., 2005; Ulrich-Vinther, 2007).

Successful engineering of teeth and the TMJ is challenging and requires the generation of functional interfaces. The introduction of BMPs in vivo to exposed pulp tissue has been proposed as a novel strategy for odontoblast transduction to enhance dentin regeneration and repair (Nakashima et al., 2006). However, gene therapy has not yet been applied to the field of total tooth engineering (Young et al., 2005; Hu et al., 2006). Engineering of the TMJ requires the creation of functional bone and cartilage with an appropriate transition zone. Investigators have generated such osteochondral grafts by seeding differentiated pig chondrocytes and Ad.BMP7-transduced human gingival fibroblasts onto biphasic PLLA/hydroxyapatite composite scaffolds and implanting them subcutaneously into N: Nih-bg-nu-xid immunocompromised mice (Schek et al., 2004, 2005). Marrow-containing vascularized bone, mature cartilage, and a defined mineralized interface can be generated within 4 wks of implantation (Schek et al., 2004, 2005). A second approach to TMJ repair is the in vivo introduction of therapeutic genes to the mandibular condyle. Recent work has demonstrated successful rAAV2-mediated transduction of VEGF to condylar tissue in vivo that subsequently enhanced mandibular condylar growth (Rabie et al., 2007) (). These pioneering studies provide proof-of-principle evidence for the fabrication of a physiologic osteochondral graft and direct TMJ transduction that may be developed to treat persons with TMJ disorders or developmental deformities.

Loss of salivary gland function can result as a pharmacologic side-effect, from radiation therapy, or as a consequence of autoimmune diseases such as Sjgrens syndrome. In addition to direct repair of non-functional glandular tissue, researchers are working to develop an engineered salivary gland substitute that could be implanted in place of the parotid gland (Aframian and Palmon, 2008). Unlike acinar cells, ductal epithelial cells are incapable of fluid secretion. Because researchers have been unable to isolate and expand acinar cells in vitro, identification and localization of membrane proteins required for ionic gradient formation and fluid flow in acinar cells have informed efforts to modify ductal cell populations by gene transfer. Acinar cells require 4 membrane proteins to generate an osmotic gradient for unidirectional fluid movement: (1) the N+K+-ATPase, used to maintain membrane potential; (2) a Ca2+-activated K+ channel; (3) the secretory isoform of the Na+/K+/2Cl- co-transporter; and (4) the apical membrane-bound Ca2+-activated Cl- channel (Melvin et al., 2005; Aframian and Palmon, 2008). Salivation occurs in response to agonists that generate an increase in intracellular Ca2+ concentration and is facilitated by osmotic gradient-directed fluid movement through water channels in the apical membrane, known as aquaporins (AQP) (Melvin et al., 2005). It is now recognized that isolated ductal epithelial cells lack expression of AQP and, as such, cannot mediate fluid movement (Tran et al., 2006). Re-introduction of transient AQP expression by adenoviral transduction has been successful in rhesus monkey parotid duct cells in vitro (Tran et al., 2005) and rat and mini-pig salivary gland tissue in vivo (Baum et al., 2006). Indeed, attempts to restore salivary flow by in vivo transduction of adenovirus encoding AQP1 into remaining glandular tissue of persons treated with radiation for head and neck cancer is the first human craniofacial repair gene therapy clinical trial and is currently ongoing (Baum et al., 2006; NIH, 2008b).

Engineering of skin and mucosal equivalents is essential for the esthetic reconstruction of individuals disfigured by trauma, resective surgery, or severe burns. Skin is composed of layered dermis and epidermis in a configuration that must be preserved for optimum regeneration. The first attempts to repair damaged skin and mucosa with an engineered graft did not occur until the 1980s (Madden et al., 1986). Skin with both dermal and epidermal components, such as DermagraftTM (Purdue et al., 1997) and ApligrafTM, used for coverage of burns and acute wounds (Eaglstein et al., 1995), was the first FDA-approved tissue-engineered construct that has been put into clinical practice. Clinically, a product known as gene-activated matrix (GAM) has been developed as an enhanced skin graft substitute. GAM for wound-specific delivery of adenovirus vector encoding PDGF-B to improve healing of diabetic ulcers is currently in Phase II clinical trials (Gu et al., 2004; NIH, 2008b). It is reasonably expected that these developments could be expanded to enhance wound healing and tissue repair in the craniofacial region (Jin et al., 2004).

Read the original post:
Gene Therapy - National Center for Biotechnology Information