Category Archives: Gene therapy

1. Two Zolgensma deaths bring gene therapy safety to spotlight  FiercePharma
2. Two Children Die After Receiving Novartis Gene Therapy  The Scientist
3. Novartis Confirms Deaths of Two Patients Treated with Gene Therapy Zolgensma  Genetic Engineering & Biotechnology News
4. Novartis reports deaths of two patients treated with Zolgensma gene therapy  BioPharma Dive
5. Novartis says two children treated with gene therapy died  Medical Xpress
6. View Full Coverage on Google News

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Two Zolgensma deaths bring gene therapy safety to spotlight - FiercePharma

As oncology practice is rapidly shifting away from toxic chemotherapy, gene therapy provides a highly specific therapeutic approach for brain tumors. This treatment is rapidly evolvingto deliver specific therapeutic genes or oncolytic viruses to eliminate the tumor, which can lead to tumor cell death and increased immune responses to tumor antigens, and disruption of the tumor microenvironment (TME), including angiogenesis/neovascularization inhibition [1]. Oncolytic virotherapy (OV), suicide gene therapy, tumor suppressor gene delivery, immunomodulatory strategies, and gene target therapies are the various types of gene therapies. Gene therapy delivery methods include direct delivery of therapeutic genes into the tumor site, which include virus-mediated adenovirus, herpes simplex virus-1 (HSV-1), adeno-associated virus-2, nonviral vector-based nanoparticles, liposomes, and micelles. Neural stem cells and mesenchymal stem cells are tumor-tropic cell carriers that express therapeutic gene(s) in the tumor site. PH-sensitive drug release, pH-sensitive liposomal carriers, and stimuli-responsive particles are examples of intelligent carriers [2].

According to the National Brain Tumor Society (NBTS), approximately 700,000 Americans have been diagnosed with a primary brain tumor, with 63% being benign and 37% being malignant. Brain tumors were the 10th leading cause of death in 2020 [3]. The pediatric brain tumors associated with neurofibromatosis type 1 (NF1) are optic pathway gliomas (OPGs), brain stem gliomas, glioblastomas, and pilocytic astrocytoma [4]. Brain and central nervous system (CNS) tumors have been reported in approximately 20% of patients with NF1 and are typically discovered in childhood. Optic pathway gliomas (OPGs) account for approximately 70% of all CNS tumors in children with NF1, while brain stem glioma accounts for approximately 17% of all CNS tumors [5]. Despite recent advances in surgery, radiotherapy, and chemotherapy, brain tumor treatment regimens have only a limited impact on long-term disease control [6]. The price of the cure is frequently unacceptable, and it includes acute and chronic organ toxicity, resistance to therapy, and more concerning, an increased risk of secondary malignancy. As a result, new strategies are required to improve overall survival and reduce treatment-related morbidity [7]. To tackle this situation, a better understanding of the functions and control of genes was needed, which paved the way for the development of gene therapy in the last decades [6].

The current study aims to provide an advance in gene therapies for pediatric brain tumors with neurofibromatosis type 1. This includes different genomic alterations seen in brain tumors and gene delivery systems comprising viral and nonviral delivery platforms along with suicide/prodrug, oncolytic, cytokine, and tumor suppressor-mediated gene therapy approaches. Finally, we discuss the results of gene therapy-mediated human clinical trials and highlight the progress, prospects, and remaining challenges of gene therapies aiming at broadeningour understanding and highlighting the therapeutic approach for pediatric brain tumors.

This systematic review was performed in March 2022 usingthe Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines [8].

Eligibility Criteria

The inclusion criteria were cases of neurofibromatosis type 1 brain tumors in all age groups with the additional inclusion of English language, free full text, articles published within the last 20 years, randomized controlled trials (RCTs), observational studies, systematic reviews, and meta-analyses. Weexcluded case reports, case studies, and editorials.

Databases and Search Strategy

The search was conducted systematically using PubMed, Cochrane, Google Scholar, and ClinicalTrials.gov databases by the first and second authors separately. Table 1 summarizes the search strategy.

The search identified a total of 1,213 articles from the databases. EndNote is used to remove duplicated articles. The remaining articleswere screened manually by the first and second authors. A total of 145 articles from databases were sought for retrieval, and 25 articles from the databaseswere retrieved and sent for quality appraisal. The articles were assessed for quality by the first two authors separately using tools depending on the study type: Cochrane Collaboration Risk of Bias Tool (CCRBT) for randomized control trials [9], Scale for the Assessment of Narrative Review Articles 2 (SANRA 2) for narrative reviews [10], and Assessment of Multiple Systematic Reviews (AMSTAR) for systematic reviews and meta-analyses [11]. Nineteen studies included in the review were scored above 70% (Figure 1).

Table 2 shows the result of the summary of the quality assessment of narrative reviews by authors.

In the study by Immonen et al., compared to controls (n = 7 patients), there is a substantial rise in the mean number of tolerated O6-benzylguanine (O6BG)/temozolomide (TMZ) cycles (P = 0.05) with gene therapy. The median progression-free survival was nine months, and the overall survival was 20 months. The study revealed delayed tumor growth at lower cumulative TMZ doses in the study patients compared to those who received standardregimens, concluding that this supports the chemoprotective effect of gene therapy when used in combination with O6BG and TMZ [25]. In the study ofAdair et al., treatment of adenovirus-mediated herpes simplex virus thymidine kinase (AdvHSV-tk) resulted in a clinically and statistically significant increase in mean survival from 39.0 19.7 (standard deviation) to 70.6 52.9 weeks (P = 0.0095). From 37.7 to 62.4 weeks, the median survival time also increased, and treatment was well tolerated. The authors concluded that AdvHSV-tk gene therapy with ganciclovir (GCV) could be a promising new treatment[26].Table 3 summarizes the risk of bias in randomized controlled trials using the Cochrane Collaboration Risk of Bias Tool (CCRBT).

Table 4 summarizes the resultof critical appraisal for systematic reviews and meta-analyses by review authors.

Brain tumors account for 21% of childhood malignancies and are the primary cause of solid tumor cancer death in children.Children affected with neurofibromatosis type 1 (NF1) are prone to optic pathway gliomas, brain stem gliomas, glioblastomas, and pilocytic astrocytoma. Two-thirds of gliomas are found in the optic pathway, with the brain stem, cerebellum, cerebral hemispheres, and subcortical structures accounting for the remaining locations. Chemotherapy is used to treat clinical progression, but surgery and radiation are difficult to use in the case of NF1-associated optic pathway gliomas since surgical resection is usually unachievable due to the tumors position. Radiation is not suggested for children with NF1 because of the possibility of secondary tumors (glioma and malignant peripheral nerve sheath tumors) in the context of this tumor predisposition syndrome, as well as the risk of late neurocognitive sequelae in children. Vincristine and carboplatin are used in first-line optic pathway glioma treatment. Vinblastine, vinorelbine, and temozolomide are the second-line chemotherapy options [28].

Overall, five-year survival rates for children less than 15 years of age are currently around 75% and 77% for those aged 15-19. Despite these advancements in treatment, a considerable number of individuals continue to be resistant to typical treatments. Acute and chronic organ damage, as well as an increased risk of secondary malignancy, are all disadvantages. Successful glioma treatment is hampered by ineffective medication distribution across the blood-brain barrier (BBB), an immunosuppressive tumor microenvironment (TME), and the development of drug resistance. Because gliomas are caused by the accumulation of genetic changes over time, gene therapy, which allows for the altering of the genetic makeup of target cells, appears to be a viable way to overcome the challenges that existing therapeuticstrategies face [7].

Figure 2 explains the pathways involved in oncogenesis. By converting the active form of guanosine triphosphate (GTP)-bound Kirsten rat sarcoma virus (KRAS) to its inactive, guanosine diphosphate (GDP)-bound state, neurofibromin directly suppresses KRAS activation. Mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are activated by GTP-bound KRAS. The activation of rapidly accelerated fibrosarcoma gene (RAF)/MAPK causes transcription and cell proliferation to increase. Unchecked KRAS activation can also result in the cross-activation of the phosphoinositide 3 kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway, which is critical for cell proliferation and survival. GTP-bound KRAS, for example, can bind and activate PI3K, resulting in survival and proliferation effects via AK strain transforming (AKT) and mTOR activity. As a result, neurofibromin deficiency can cause disease in a variety of ways. In gliomas, the KRAS, PI3K/phosphatase and tensin homolog (PTEN)/AKT pathways and neurogenic locus notch homolog protein (NOTCH) signaling are linked to cancer cell proliferation[29].

Glioblastoma Multiforme (GBM)

Complete resection of GBM is virtually impossible due to its intrusive nature and sensitive location. The current standard of care is a maximum surgical resection followed by radiation and temozolomide chemotherapy; however, the median survival time is still fewer than 15 months. This necessitates the creation of gene therapy, which delivers oncolytic viruses to the tumor in a precise manner to destroy it and lead to tumor cell death as well as increased immune responses to tumor antigens and disturbance of the tumor microenvironment, including angiogenesis/neovascularization inhibition [1].The common gene targets that are mutated or upregulated in glioblastoma are neurofibromin, epidermal growth factor receptor (EGFR), phosphate and tensin (PTEN) homolog, platelet-derived growth factor (PDGF) receptor-alpha, isocitrate dehydrogenase-1 (IDH1), and tumor suppressor p53. GBM is a suitable candidate for gene therapy for several reasons:tumors remain within the brain with only rare metastases to other tissues; most cells in the brain are postmitotic, which allows for precise targeting of dividing tumor cells; tumors are often accessible neurosurgically for vector administration; sophisticated imaging paradigms are available; and standard therapies are minimally successful.

Delivery Methods for Gene Therapy

Table 5 summarizes the advantages, limitations, and clinical trials of the viral vectors used for gene therapy.

Table 6 summarizes the advantages and limitationsof the nonviral vectors used for gene therapy.

Table 7 summarizes the advantages and limitations of tumor-tropic cell carriers expressing therapeutic gene(s) in the tumor site.

Oncolytic Virotherapy (OV)

OVs are intended to particularly infect cancer cells, self-replicate, induce oncolysis, and amplify therapeutic genes at tumor sites [27]. The advantages of OV include its high transduction efficiency and intra-tumoral viral spread, the capability of producing high viral titers, accessibility to genetic engineering, and adding additional therapeutic transgenes. Its limitations include host immune rejection/suppression of the virus, safety risks surrounding replication-competent viruses, and requirement of local administration during surgery[30]. Figure 3 explains the mechanism of action of oncolytic virotherapy.

Oncolytic herpes simplex virus (oHSV) are double-stranded deoxyribonucleic acid (DNA) viruses, a human pathogen with neurotropic properties. The challenge in designing oHSVs is to provide tumor selectivity while maintaining an acceptable safety profile [27]. Early clinical trial results showed that numerous oHSV vectors had high safety profiles with no signs of encephalitis but poor therapeutic effectiveness [31].

Conditionally replicating adenovirus (CRAd) are nonenveloped DNA viruses capable of infecting both the dividing and nondividing cells. An important advantage of CRAd viruses is that they are naturally non-neurotropic and have an enhanced safety profile over the oHSV vector. ONYX-015 and Ad5-Delta24 bare the most widely studied CRAd [14]. ONYX-015 contains a deletion in the viral protein early region 1B-55K (E1B-55K), which normally binds to and inactivates the host cell p53 protein. Therefore, it is assumed that cells with functional p53 cannot support viral replication in the absence of this protein, whereas tumor cells with a nonfunctional support viral replication.

Oncolytic measles, reovirus vectors, and recombinant nonpathogenic polio rhinovirus (PVS-RIPO) are reoviruses that only replicate in glioma cells because platelet-derived growth factor receptor (PDGFR) or EGFR stimulation of the KRASpathway suppresses ribonucleic acid (RNA)-activated protein kinase activation. Clinical trial demonstrates that they are safe and well-tolerated with no evidence of clinical encephalitis. Measles virus (MV) exhibits the mutated hemagglutinin envelope glycoprotein H, which targets the cluster of differentiation 46 (CD46) on glioma cells. The circulating carcinogenic embryonic antigen (CEA) was modified into MV, which can be used to measure virus replication and oncolytic function [27]. PVS-tumor RIPOs cell tropism is determined by the poliovirus receptor CD155, which is expressed on high-grade glioma cells. The clinical trialsfindings revealed satisfactory antitumor effectiveness but a low safety profile. Table 8 summarizes clinical trials and results on oncolytic virotherapy.

Suicide Gene Therapies

The suicide gene technique is based on virally delivering suicide genes to target cells, which produce enzymes that convert prodrugs to active compounds. The inert prodrug is given systematically and then activated by suicide enzymes at the tumor site, resulting in tumor cell apoptosis [27]. Its advantages include achieving a bystander effect, requiring short-term gene expression, selective tumor cell targeting, and enhancing sensitivity to conventional therapy. It is restricted by the limited spatial distribution of gene transfer vectors, poor gene transfer efficiency into tumor cells in vivo, inability to target dispersed tumor cells, and restricted intra-tumoral distribution.Figure 4 explains the mechanism of action of suicide gene therapy.

Herpes simplex virus thymidine kinase (HSV-tk) enzyme catalyzes ganciclovir/valacyclovir monophosphorylation, which occurs after the triphosphorylation and activation of intracellular kinases. The active medication inhibits DNA synthesis and tumor lysis by blocking the S phase and arresting the cell circle. Cytosine deaminase (CD) catalyzes the activation of the prodrug 5-fluorocytosine (5-FC). A replication-competent retrovirus called Toca 511 loads the CD and transinfects tumor cells. It stimulates the expression of CD, which activates the 5-FU, which blocks DNA synthesis irreversibly and causes cell death. Escherichia coli-derived purine nucleoside phosphorylase (PNP)transforms adenosine ribonucleosides, such as fludarabine, into the active adenine molecule, 2-fluoroadenine, which disrupts RNA replication and the cell cycle. Antibiotic therapy, which suppresses intestinal flora, may over-activate the PNP gene therapy, resulting in increased prodrug conversion [27]. Table 9 summarizes clinical trials and results on suicide gene therapies.

Tumor Suppressor Gene Therapies

High-grade gliomas frequently have deletions and mutations in tumor suppressor genes such as p53, p16, and phosphatase and tensin homologs (PTEN) [2]. Tumor suppressor gene techniques aim to restore normal function by transferring antitumoral functional genes to glioma cells. The advantages are safety in clinical trials, the potential to induce senescence within tumors, and the potential to sensitize tumor cells to other therapies. The limitations are as follows: multiple redundant pathways in tumors hinder efficacy, poor in vivo gene transfer, and limited distribution of therapy.Figure 5 explains the mechanism of action of tumor suppressor gene therapy.

P53 is involved in the inhibition of angiogenesis and DNA repair pathways. E1 gene is replaced by wild-type p53 in adenovirus and transmitted via a cytomegalovirus promoter (Ad5CMV-p53), which is the most widely used method. The E1 deletion prevents the virus from starting the infectious phase, while the cytomegalovirus promoter boosts the production of the p53 gene [27].

P16 prevents uncontrolled replication and oncogenesis by arresting the cell cycle during the G1-S transition [32]. Restoration of p16 function through an adenoviral vector has been found to decrease glioma growth and locoregional dissemination while also inhibiting matrix metalloprotease activity in the glioma microenvironment [33]. The adenovirus-mediated p16 gene was used to drive p16-null human glioma cell lines to enter phase G1 of the cell cycle. In HGG cells, data revealed that p16 expression is linked to tumor radiosensitivity through mechanisms of aberrant nucleation [34]. It is worth noting that the efficiency of the p16 gene approach is contingent on maintaining retinoblastoma protein (pRB) activity [35].

The PTEN gene has been shown to suppress glioma proliferation and induce oncolysis when delivered through an adenoviral vector [27]. Adenoviral vector transfer of the PTEN gene into glioma cells improved tumor sensitivity to temozolomide and radiation [36].Table 10 summarizes clinical trials and results on tumor suppressor gene therapies.

Immunomodulatory Gene Therapies

The objective of anti-glioma immunomodulatory gene therapy is to induce or augment the T-cell-mediated immune response against tumors using the delivery of genes for immunostimulatory cytokines and interferon beta/gamma (IFN-/) [27]. Its advantages include the following: this therapy can achieve passive or active tumor immunity, it has the possibility to eliminate tumor cells that remain post-surgery, and it regulates the tumor microenvironment. This therapy is limited by tumor-induced immunosuppression, lack of antigen-presenting dendritic cells within the brain, and overcoming the presence of immune-suppressive regulatory T-cells and cytokines.Figure 6 explains the mechanism of action of immunomodulatory gene therapy.

The stimulation of natural killer cells and macrophages demonstrated potential antitumoral action [37]. INF- was also transferred using nanoparticles and liposomes. Clinical trial shows a reduction in volumetric glioma and mild toxicity [38]. Histological findings reported an elevated level of immune activation[39]. IFN- inhibits cancer cell proliferation and interactions with the extracellular matrix [40].

Interleukin-12 (IL-12) is one of the most important immunostimulant cytokines for strengthening the immune system and attracting cytotoxic cells in the tumor microenvironment. Nonreplicating adenoviruses and HSV were used in an earlier phase of research to deliver IL-12 to malignant glioma cells. Preclinical research revealed tumor cell death, active microglia cell infiltration, a favorable safety profile, and a significant local immune response [27].

Several clinical trials have shown that chemotherapy has a synergistic impact when combined with immunotherapy, challenging the conventional dogma that chemotherapy-induced immunosuppression prevents the formation of antitumor immune responses. In a limited phase I clinical trial, three pediatric patients with recurring brain tumors were given a combination of high-dose chemotherapy and adoptive immunotherapy [41]. Accumulating preclinical and clinical evidence suggests that combining tumor cell killing techniques with immunotherapy results in synergism between the two therapies, resulting in improved efficacy and lower toxicity. This collection of evidence refutes the conventional notion that tumor cell killing tactics hinder the immune systems ability to recognize and eradicate a brain tumor, and it supports the use of combined cytotoxic-immunotherapeutic strategies in the treatment of glioblastoma multiforme patients [42].Table 11 summarizes clinical trials and results on immunomodulatory gene therapy.

Gene Target Therapies

Gene target medicinesdirectly bind specific tumor antigens to block oncogenic pathways irreversibly.Figure 7 explains the target gene mechanism of action.

The epidermal growth factor receptor (EGFRvIII) variation, which is prevalent in 30% of high-grade gliomas, is involved in oncogenesis and tumor development processes. Antisense or short interfering RNA (siRNA) directed exclusively targeting the thymidine kinase domain of glioma EGFRvIII was delivered by viral vectors and nanoparticles [43]. The delivery of EGFRvIII siRNA using cyclodextrin-modified dendritic polyamine complexes (DexAMs) exhibited promising effects in malignant glioma cells, even when combined with erlotinib [44].

Direct intra-tumoral inoculation of polyethylenimine (PEI)/VEGF siRNA had a substantial antiangiogenic impact on xenografts [44]. In the Matrigel plug experiment, Ad-DeltaB7-shVEGF, an adenovirus construct, was developed, expressing a short hairpin RNA against VEGF; it showed excellent antiangiogenic action and better bioavailability than replication-incompetent adenoviruses [45]. In a human xenografted glioma model, Ad-DeltaB7-KOX,an oncolytic adenovirus, showed strong anticancer efficacy [46]. Another study looked at HGGs infected with adenovirus expressing vascular endothelial growth factor receptor (VEGFR) and the oncolytic virus dl922/947. This combination therapy was more successful than monotherapy [27].

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Systematic Review of Pediatric Brain Tumors in Neurofibromatosis Type 1: Status of Gene Therapy - Cureus

DUBLIN, Aug. 11, 2022 /PRNewswire/ --The "Global Gene Therapy Market by Vectors (Non-viral(Oligonucleotides), Viral(Retroviral, Adeno-associated)), Indication (Cancer, Neurological, Hepatological Diseases, Duchenne Muscular Dystrophy), Delivery Method (In Vivo, Ex Vivo), and Region - Forecast to 2027" report has been added to ResearchAndMarkets.com's offering.

The global gene therapy market is valued at an estimated USD 7.3 billion in 2022 and is projected to reach USD 17.2 billion by 2027, at a CAGR of 18.6% during the forecast period. Factors such as rising cases of neurological diseases and cancer, growing gene therapy product approvals, and increasing investment in gene therapy related research and development drive the market growth. However, factors like high cost of gene therapy is restraining the growth of this market.

The cancer segment accounted for the highest growth rate in the gene therapy market, by indication, during the forecast period

In 2021, cancer segment accounted for the highest growth rate. Growing disease burden of cancer across the globe coupled with rising demand for gene therapies to treat cancer will augment the segmental growth of cancer over the forecast period.

Asia-Pacific: The fastest-growing region in the gene therapy market

The Asia-Pacific market is estimated to record the highest CAGR during the forecast period. The high growth rate of this market can be attributed to the improving healthcare expenditure in emerging economies, increasing product launches, and increasing incidence of cancer and neurological diseases.

Research Coverage

This report provides a detailed picture of the global gene therapy market. It aims at estimating the size and future growth potential of the market across different segments such as vectors, indication, delivery method, and region. The report also includes an in-depth competitive analysis of the key market players along with their company profiles recent developments and key market strategies.

List of Companies Profiled in the Report

Premium Insights

Market DynamicsDrivers

Opportunities

Challenges

Key Topics Covered:

1 Introduction

2 Research Methodology

3 Executive Summary

4 Premium Insights

5 Market Overview

6 Gene Therapy Market, by Vector

7 Gene Therapy Market, by Indication

8 Gene Therapy Market, by Delivery Method

9 Gene Therapy Market, by Region

10 Competitive Landscape

11 Company Profiles

For more information about this report visit https://www.researchandmarkets.com/r/x356k5

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Global Gene Therapy Market to Grow from $7.3 Billion to $17.2 Billion by 2027 - PR Newswire

Published: Published Date - 09:21 PM, Wed - 10 August 22

Hyderabad: This concept may seem quite fictional and even futuristic. However, this is what geneticists worldwide through gene therapy are pursuing, while trying to find cure for a wide range of diseases that challenge modern medicine including cancers, heart diseases, diabetes, haemophilia, AIDS, genetic disorders, among others.

Gene therapy involves altering the genes inside the cells of the human body, in order to treat or prevent the disease progression. Essentially, geneticists worldwide are exploring ways to utilise gene therapy to alter genetic composition of cells that are responsible for causing diseases and in the process find a long term cure for diseases. The potential to unlock the cure for a wide range of diseases has become a major driving force for researchers and pharma giants worldwide to focus their energies and resources on gene therapy.

So what exactly is gene and gene therapy?

The Gene Therapy Advisory and Evaluation Committee (GTAEC), which monitors clinical trials across India on gene therapies, defines Gene is the most basic and functional unit of heredity and inheritance and consists of a specific sequence of nucleotides in DNA or RNA located on chromosomes that encodes for specific proteins. The human genome comprises more than 20,000 genes. Gene therapy refers to the process of introduction, removal or change in content of an individuals genetic material with the goal of treating the disease and a possibility of achieving long term cure.

The genetic material that has to be introduced to the diseased cell is done through a vector, whch is usually a virus. Viruses are the preferred vectors or vehicles as they are adaptable and efficient in delivering genetic material, the GTAEC, said.

While worldwide major pharmaceutical companies are developing gene therapies for treatment of single gene defects like haemophilia and muscular dystrophy, the Department of Biotechnology (DBT), Government of India, Tata Memorial Hospital, Mumbai and IIT-Mumbai have collaborated to start clinical trials of gene therapy on cancer in India.

Gene therapy in cancer:

In the last few years, CAR- (Chimeric Antigen Receptor) T therapy, a form of gene therapy has emerged as a breakthrough treatment for cancer, especially for leukemia, lymphoma (cancer of the lymphatic system) and multiple myeloma or the cancer of the plasma cells.

The CAR-T cells are genetically engineered in a laboratory and they bind with the cancer cells and kill them. The therapy is available in a few cancer research centres (on clinical trials basis) in US and cost of treatment ranges anywhere from Rs 3 crore to Rs. 4 crore.

To reduce treatment costs, promote and support development CAR-T cell technology against cancers, for the first time in India, Biotechnology Industry Research Assistance Council (BIRAC), established by DBT, Tata Memorial Hospital and IIT Bombay, have launched clincal trials of CAR-T gene therapy to treat cancers. The CAR-T cells were designed and manufactured at Bioscience and Bioengineering (BSBE) department of IIT Bombay. The gene therapy study on cancers is in early phase clinical trials at Tata Memorial in Mumbai.

Regulation of gene therapy:

Realising the potential of gene therapies in treating complex diseases, the GOI is providing financial and even technical guidance to researchers through ICMR, DBT and DST. To ensure gene therapies are introduced in India and clinical trials for gene therapies are performed in an ethical, scientific and safe manner, the ICMR has also framed National Guidelines for Gene Therapy Product Development and Clinical Trials document.

Excerpt from:
Health and Tech: The promise of gene therapy to cure cancers - Telangana Today

Cell culture and cell lines

The mouse cell lines MCA205 H-2b (MCA, methylcholanthrene derived sarcoma, provided by Dr. Guido Kromer, France) and B16F10 (B16, melanoma, kindly provided by Dr. Roger Chammas, ICESP) were maintained in a humidified incubator at 37C with 5% CO2 and cultivated in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Invitrogen) as well as 1X Anti-Anti (AntibioticAntimycotic -100X, Thermo Fisher Scientific). HEK293 cells were cultivated in Dulbeccos modified Eagle medium (both from Thermo Fisher Scientific), supplemented and maintained in the same conditions as above.

Here we use the MCA sarcoma cell line and employed an intratumoral (i.t) application model since it was demonstrated under these conditions the ability of Dox to unleash ICD and stimulate immune responses in vivo11. We also used the B16 cell line, as it was with this model that we revealed the cell death and immune stimulatory events of our p19Arf/IFN treatment. With regard to the treatment order, we based our approach on the work of Fridlender and collaborators (2010) that showed that association of an adenoviral vector encoding IFN with chemotherapy is more effective when gene transfer is applied first23.

The MCA-DEVD cell line was generated by transduction with a lentivirus reporter for caspase-3 activity and selection for puromycin resistance (0.5g/ml). This vector, previously described24, encodes a constitutively expressed luciferase-GFP protein separated from a polyubiquitin domain via a caspase-3 cleavage site and was generously provided by Dr. Chuan-Yuan Li (Department of Radiation Oncology, University of Colorado School of Medicine, Aurora, CO, USA).

Construction and production of AdRGD-PG adenoviral vectors (serotype 5) containing modification with the RGD motif in the fiber as well as the p53-responsive promoter (PGTx, PG) has been described previously14. Titration of adenoviral stocks was performed using the Adeno-X Rapid Titer Kit (Clontech, Mountain View, CA, USA) and titer yields were: AdRGD-CMV-LacZ (3.6109IU/mL, infectious units/milliliter), AdRGD-PG-LUC (11011IU/mL), AdRGD-PG-eGFP (51010IU/mL), AdRGD-PG-p19 (1.31010IU/mL) and Ad-RGD-IFN (51010IU/mL). This biological titer was used to calculate multipilicity of infection (MOI).

MCA or B16 cells (1105) were plated in 6 well plates containing 1mL of RPMI media and transduced with adenovirus at the desired MOI. After an overnight transduction period (1216h), 2mL of media was added and cells kept in culture until needed. When combining adenoviral transduction with chemotherapy, Dox (doxrubicin hydrochloride, Sigma, St. Louis, MO, USA) was added immediately after the overnight transduction using the concentration indicated for each experiment. Importantly, in the Dox single treatment condition, Dox was added at the same moment as in the association group, 12 to 16h after cell plating. After 12h treatment with Dox (1mg/mL) or Nutlin-3 (10M, Sigma), expression of eGFP from AdRGD-PG-eGFP was analyzed by flow cytometry (Attune, Life Technologies). Cell viability was assessed by MTT assay where, 8h after transduction in 6 well plates, 2104 cells/well were plated in 96 well plates, treated with Dox, and analyzed after 16h of incubation. Non-transduced cells were used as viable control and protocol was carried out as described previously25. Cell cycle analysis by propidium iodide (PI) staining was carried out 72h after p19Arf/IFN and Dox single treatment, as previously described16. Analysis of caspase 3 activity in vitro was performed 16h after combined treatment using the CellEvent Caspase-3/7 Green Reagent (Thermo Fisher Scientific) by flow cytometry, following manufacturers instructions. Last, analysis of ICD markers upon p19Arf/IFN+Dox was conducted as detailed previously14. Briefly, detection of calreticulin+ and PI- cells was made 14h after combined treatment, by staining with a CRT-specific antibody (1:100, Novus, Biologicals, CO, USA) and after cells were washed with PBS, they were incubated with Alexa488-conjugated anti-rabbit secondary antibody (1:500, Thermo Fisher Scientific) followed by PI staining to exclude dead cells, immediately before flow cytometry. Accumulation of ATP in the cell supernatant was detected using the ENLITEN ATP Assay (Promega, Madison, WI, USA), as per the manufacturer's instructions. Luminescence was observed using a GloMax Plate Reader (Promega). HMGB1 in cell supernatant was detected by Western blot after conditioned medium was supplemented with protease inhibitor cocktail (Thermo Fisher Scientific). Then, 180l of the medium was concentrated (Concentrator PlusEppendorf, Hamburg, Germany) and subjected to western blotting. Unrelated, high molecular weight regions of the membrane were removed before detection was performed using anti-HMGB1 (Abcam ab79823, Cambridge, UK) and a secondary antibody conjugated with horseradish peroxidase before visualization using ECL (GE Healthcare, Chicago, IL, USA) and the ImageQuant LAS4000 imaging platform (GE Healthcare). See Supplementary Information Westerns S2 for original images from three independent assays. Additional Western blots were performed using cell lysates, high-molecular weight regions of the membranes were removed and then detection was performed using anti- PARP (Cell Signaling, Danvers, MA, USA, #9542), anti-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, #47778), anti-Caspase 3 (Cell Signaling, #9662), anti-Tubulin (Millipore, Burlington, MA, USA, #05-829) and the appropriate secondary antibodies conjugated with horseradish peroxidase (anti-mouseSigma #A9044 e anti-rabbitSigma #A0545). See Supplementary Information Westerns S2 for original images from two independent assays.

The influence of two independent variables, namely, MOI of adenoviral vectors encoding p19Arf/IFN and the concentration of Dox, was investigated on MCA and B16 cells using factorial experiments in five levels (Table S1), with the percentage of hypodiploid cells as the variable response. The experiments were carried out employing central composite rotational design (CCRD) where, for each cell line, a set of twelve combinatory assays containing a central composite factorial matrix plus rotation points, central points and controls was performed (Table S2, where the assays and conditions are provided in detail). To better visualize the effects and interactions of MOI and Dox concentration on the percentage of hypodiploid cells, assessed by PI staining after 20h of treatment, the results were plotted in response surface graphs.

Importantly, the statistical significance of the independent variables and their interactions was determined by Fishers post-test for an analysis of variance (ANOVA) and Pareto chart analysis, both at a confidence level of 95% (p0.05). Moreover, five repetitions at the central point (CP) assays were used to minimize the error term of the ANOVA. Experimental designs, data regression and graphical analysis were performed using the Statistica software v.7.0 (Statsoft, Inc., Tulsa, OK, USA).

Both C57BL/6 and Nude mice were female, 7weeks old, obtained from the Centro de Bioterismo da FMUSP and kept in the animal facility in the Centro de Medicina Nuclear (CMN) in SPF conditions, with food and water ad libitum. The methods are reported in accordance with ARRIVE guidelines. The well-being of the mice was constantly monitored and all methods, including vaccination protocols, in vivo gene therapy, imaging, echocardiographic assessments, anesthesia and euthanasia were carried out in accordance with relevant guidelines and regulations of Brazil and our institution whose ethics committee (Committee for the Ethical Use of Animals, CEUA, University of So Paulo School of Medicine, FMUSP) approved this project (protocol n 165/14).

In the first step of the immunotherapy model, nave C57BL/6 mice were inoculated (s.c) in the right flank (tumor challenge site) with fresh untreated MCA (2105) or B16 (6104) cells and in the second step, vaccinated (s.c) on days+3,+9 and+15 with 3105 ex vivo treated cells applied in the left flank (vaccine site). Ex vivo treatment was carried out as follows: MCA or B16 cells were seeded in 10cm plates with 2mL of media and co-transduced with the AdRGD-PG-p19 and AdRGD-PG-IFN (MOI 500 for each) for 4h before the addition of 8mL of fresh media. Then, cells were kept in culture for 16h and in the p19Arf/IFN+Dox or Dox groups, Dox (14M) was added for 6h, until cells were harvested, washed twice with cold PBS, counted and resuspended in 100 L of cold PBS. For the DEAD cell+GFP control group, cells were transduced with the AdRGD-PG-eGFP vector (MOI 1000) and after 16h, harvested, washed twice with cold PBS, resuspended and lysed by three cycles of freezing and thawing.

MCA (2105) or B16 (5105) cells were harvested, washed twice with cold PBS, resuspended in 100 L of PBS per mouse and then inoculated subcutaneously (s.c) in the left flank of immune competent C57BL/6 or immune deficient Balb/c Nude (Foxn1n) mice. While mice were not randomized after injection of cells, but there was no specific selection of animals for each treatment group. No blinding of group allocation was performed at any phase of experimentation. No animals were excluded from the data. Approximately 8days later, palpable (60 mm3) tumors were treated three times, once every 2days, with intratumoral (i.t) injections (administered with precision Hamilton glass syringes (volume 100L) and 26G needles) of the following adenoviral vectors, AdRGD-CMV-LacZ or AdRGD-PG-LUC (4108IU, resuspended in 25 L final volume of PBS/mouse) or treated with the combination of AdRGD-PG-p19 and AdRGD-PG-IFN (2108IU, for each vector and maintaining the 25 L final volume per mouse). For the Dox single treatment model, chemotherapy was applied (i.t) once on day 12 with the following doses: 60, 20, 10 or 5mg/kg (in the final volume of 30 L of PBS/mouse). Whereas in the association model, adenoviral vectors were injected as explained above and Dox given 2days after the last viral injection (day 14), following the injection method as the Dox single treatment group. Tumor progression was measured by calipers every two days and volume calculated as described17. For the survival analysis comparing C57BL/6 and Nude mice, treated mice were euthanized by anesthesia with ketamine/xylazine followed by CO2 inhalation when tumor volume reached 1000 mm3 unless otherwise noted. See figure legends for the number of animals in each experimental group.

For the analysis of caspase 3 in vivo, MCA-DEVD tumors were treated in situ as described above and 24 and 48h after the last treatment injection, mice were submitted to bioluminescence imagining (IVIS Spectrum, Caliper Life Science) to detect the luciferase activity from the DEVD reporter. To this end, 10mg/kg luciferin (Promega) was administered by intraperitoneal (i.p) injection of each mouse and these were anesthetized with isoflurane (Cristalia, So Paulo, Brazil) using the Xenogen anesthesia system before imaging. Images were captured and only the strongest signal from each tumor was included in the analysis with Living Imaging 4.3 software (PerkinElmer, Waltham, MA, USA). Luciferase activity was obtained from the average radiance value [p/s/cm2/sr]. To calculate the fold activity overtime, average radiance values obtained for each mouse 48h post-treatment were divided by its respective value at 24h. Parental MCA tumors were used as negative control and no emission was detected (data not shown).

The systolic cardiac function was assessed by echocardiography. Exams were performed 10days after treatments with AdRGD-PG-eGFP (adenovirus control), Dox 10mg/kg, Dox 20mg/kg and p19Arf/IFN+Dox 10mg/kg. Mice were anesthetized with 1.5 to 2.5% isoflurane (in 100% oxygen ventilation). They were trichotomized and placed in supine decubitus to obtain cardiac images. Parasternal-long and short axis images were captured using VEVO 2100 ultrasound equipment (Vevo 2100 Imaging System, VisualSonics, Toronto, Canada) with a 40MHz linear-transducer. Analyses were performed off-line using VevoCQ LV Analysis software (VisualSonics). Parameters such as systolic and diastolic volumes were calculated using Simpsons modified algorithms present in the analysis software (parasternal-long axis images). Based on these volumes, stroke volume (L) and left ventricle ejection fraction (LVEF, %) were calculated. Also, linear measurements were obtained from parasternal short axis images. Left ventricle shortening fraction (LVSF, %) was calculated, using systolic and diastolic diameters. Left ventricle mass (LV mass, mg) was estimated by linear measurements. Beating rate (beats per minute, BPM) was recorded directly by an animal table-ECG system connected to the VEVO 2100 system. Echocardiographic results were interpreted considering the American Society of Echocardiography recommendations concerning the mouse model26. All parameters were shown as the mean values of three consecutive cardiac cycles. Transthoracic echocardiography image acquisition and analysis was performed by an expert investigator who was blind to the experimental groups.

Data are presented as meanSEM. Statistical differences between groups are indicated with p values, being *p<0.05, **p<0.01 and ***p<0.001. Statistical tests are indicated in each figure legend along with the number of independent experiments performed or number (n) of mice in each group. These analyses were made using the GraphPad Prism 5 (La Jolla, CA, USA) software, with the exception of the CCRD analysis (explained above).

Continued here:
Potentiation of combined p19Arf and interferon-beta cancer gene therapy through its association with doxorubicin chemotherapy | Scientific Reports -...

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

SOUTH SAN FRANCISCO, Calif.--(BUSINESS WIRE)--Graphite Bio, Inc. (Nasdaq: GRPH), a clinical-stage, next-generation gene editing company harnessing the power of high-efficiency precision gene repair to develop therapies with the potential to cure serious diseases, today announced that the first patient has been dosed with GPH101, now called nulabeglogene autogedtemcel (nula-cel), in the companys Phase 1/2 CEDAR trial in people with sickle cell disease (SCD). Nula-cel is an investigational gene editing therapy designed to directly correct the genetic mutation that causes SCD and definitively cure the disease.

For decades, the goal of gene editing has been to precisely correct genetic mutations that cause disease. Today, we took an important step toward achieving that goal by dosing our first patient with nula-cel, the first investigational therapy designed to correct a mutated gene to normal. This first use of high-efficiency precision DNA repair to correct a genetic mutation is an important milestone not only for our company but also for the gene editing field and, hopefully, for the sickle cell community, said Josh Lehrer, M.D., M.Phil., chief executive officer of Graphite Bio.

We continue to make tremendous progress with the development of nula-cel, which in preclinical studies successfully corrected the sickle cell disease mutation, directly reducing sickle hemoglobin and restoring healthy adult hemoglobin to potentially curative levels, Lehrer continued. We believe nula-cel could be a definitive cure for sickle cell disease, with the potential to address all complications associated with this life-threatening disease. We look forward to reporting initial proof-of-concept data from the CEDAR trial in mid-2023.

The CEDAR trial is a Phase 1/2 open-label, single-dose clinical trial evaluating the safety, preliminary efficacy and pharmacodynamics of nula-cel in approximately 15 patients with severe SCD. The trial is currently enrolling patients at multiple sites in the United States.

About nula-cel

Nula-cel, formerly known as GPH101, is an investigational next-generation gene-editing autologous hematopoietic stem cell (HSC) therapy designed to directly correct the genetic mutation that causes sickle cell disease (SCD). A serious, life-threatening inherited blood disorder, SCD affects approximately 100,000 people in the United States and millions of people around the world, making it the most prevalent monogenic disease worldwide. Nula-cel is the first investigational therapy to use a highly differentiated gene correction approach that seeks to efficiently and precisely correct the mutation in the beta-globin gene to decrease sickle hemoglobin (HbS) production and restore adult hemoglobin (HbA) expression, thereby potentially curing SCD. The U.S. Food and Drug Administration (FDA) granted Fast Track and Orphan Drug designations to nula-cel for the treatment of SCD.

Graphite Bio is evaluating nula-cel in the CEDAR trial, an open-label, multi-center Phase 1/2 clinical trial designed to assess safety, engraftment success, gene correction rates, total hemoglobin, as well as other clinical and exploratory endpoints and pharmacodynamics in patients with severe SCD.

About Graphite Bio

Graphite Bio is a clinical-stage, next-generation gene editing company driven to discover and develop cures for a wide range of serious and life-threatening diseases. The company is pioneering a precision gene editing approach that has the potential to transform human health by achieving one of medicines most elusive goals: to precisely find & replace any gene in the genome. Graphite Bios UltraHDR gene editing platform takes CRISPR beyond cutting and harnesses the power of high-efficiency precision DNA repair, also known as homology directed repair (HDR), to precisely correct genetic mutations, replace entire disease-causing genes with functional genes or insert new genes into predetermined, safe locations. The company was co-founded by academic pioneers in the fields of gene editing and gene therapy, including Maria Grazia Roncarolo, M.D., and Matthew Porteus, M.D., Ph.D.

Learn more about Graphite Bio by visiting http://www.graphitebio.com and following the company on LinkedIn and Twitter.

Forward-Looking Statements

Statements we make in this press release may include statements which are not historical facts and are considered forward-looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended (the Securities Act), and Section 21E of the Securities Exchange Act of 1934, as amended (the Exchange Act). These statements may be identified by words such as aims, anticipates, believes, could, estimates, expects, forecasts, goal, intends, may, plans, possible, potential, seeks, will, and variations of these words or similar expressions that are intended to identify forward-looking statements. Any such statements in this press release that are not statements of historical fact, including statements regarding our nula-cel (formerly GPH101) product candidate, its clinical and therapeutic potential, our plans to advance nula-cel in our Phase 1/2 CEDAR trial and to report initial proof-of-concept data, and the timing of these events, may be deemed to be forward-looking statements. We intend these forward-looking statements to be covered by the safe harbor provisions for forward-looking statements contained in Section 27A of the Securities Act and Section 21E of the Exchange Act and are making this statement for purposes of complying with those safe harbor provisions.

Any forward-looking statements in this press release are based on Graphite Bios current views about our plans, intentions, expectations, strategies and prospects only as of the date of this release and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements, including the risk that we may encounter regulatory hurdles or delays, for example, in patient enrollment and dosing, and in the progress, conduct and completion of our Phase 1/2 CEDAR trial and our other planned clinical trials. These risks concerning Graphite Bios programs and operations are described in additional detail in its periodic filings with the SEC, including its most recently filed periodic report, and subsequent filings thereafter. Graphite Bio explicitly disclaims any obligation to update any forward-looking statements except to the extent required by law.

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Graphite Bio Doses First Patient with Investigational Gene Editing Therapy GPH101 for Sickle Cell Disease - Business Wire

DUBLIN--(BUSINESS WIRE)--The "Stem Cell Therapy Global Market Opportunities And Strategies To 2031" report has been added to ResearchAndMarkets.com's offering.

The global stem cell therapy market reached a value of nearly $4,019.6 million in 2021, having increased at a compound annual growth rate (CAGR) of 70.9% since 2016. The market is expected to grow from $4,019.6 million in 2021 to $10,600.2 million in 2026 at a rate of 21.4%. The market is then expected to grow at a CAGR of 11.4% from 2026 and reach $18,175.4 million in 2031.

Growth in the historic period in the stem cell therapy market resulted from rising prevalence of chronic diseases, a rise in funding from governments and private organizations, rapid growth in emerging markets, an increase in investments in cell and gene therapies, surge in healthcare expenditure, and an increase in pharmaceutical R&D expenditure. The market was restrained by low healthcare access in developing countries, limited reimbursements, and ethical concerns related to the use of embryonic stem cells in the research and development.

Going forward, increasing government support, rapid increase in the aging population, rising research and development spending, and increasing healthcare expenditure will drive market growth. Factors that could hinder the growth of the market in the future include high cost of stem cell therapy, stringent regulations imposed by regulators, and high cost of storage of stem cells.

The stem cell therapy market is segmented by type into allogeneic stem cell therapy and autologous stem cell therapy. The autologous stem cell therapy segment was the largest segment of the stem cell therapy market segmented by type, accounting for 100% of the total in 2021.

The stem cell therapy market is also segmented by cell source into adult stem cells, induced pluripotent stem cells, and embryonic stem cells. The induced pluripotent stem cells was the largest segment of the stem cell therapy market segmented by cell source, accounting for 77.2% of the total in 2021. Going forward, the adult stem cells segment is expected to be the fastest growing segment in the stem cell therapy market segmented by cell source, at a CAGR of 21.7% during 2021-2026.

The stem cell therapy market is also segmented by application into musculoskeletal disorders and wounds & injuries, cancer, autoimmune disorders, and others. The cancer segment was the largest segment of the stem cell therapy market segmented by application, accounting for 49.7% of the total in 2021. Going forward, musculoskeletal disorders and wounds & injuries segment is expected to be the fastest growing segment in the stem cell therapy market segmented by application, at a CAGR of 22.1% during 2021-2026.

The stem cell therapy market is also segmented by end-users into hospitals and clinics, research centers, and others. The hospitals and clinics segment was the largest segment of the stem cell therapy market segmented by end-users, accounting for 66.0% of the total in 2021. Going forward, hospitals and clinics segment is expected to be the fastest growing segment in the stem cell therapy market segmented by end-users, at a CAGR of 22.0% during 2021-2026.

Scope:

Markets Covered:

Key Topics Covered:

1. Stem Cell Therapy Market Executive Summary

2. Table of Contents

3. List of Figures

4. List of Tables

5. Report Structure

6. Introduction

7. Stem Cell Therapy Market Characteristics

8. Stem Cell Therapy Trends And Strategies

9. Impact Of Covid-19 On Stem Cell Therapy Market

10. Global Stem Cell Therapy Market Size And Growth

11. Global Stem Cell Therapy Market Segmentation

12. Stem Cell Therapy Market, Regional And Country Analysis

13. Asia-Pacific Stem Cell Therapy Market

14. Western Europe Stem Cell Therapy Market

15. Eastern Europe Stem Cell Therapy Market

16. North America Stem Cell Therapy Market

17. South America Stem Cell Therapy Market

18. Middle East Stem Cell Therapy Market

19. Africa Stem Cell Therapy Market

20. Stem Cell Therapy Global Market Competitive Landscape

21. Stem Cell Therapy Market Pipeline Analysis

22. Key Mergers And Acquisitions In The Stem Cell Therapy Market

23. Stem Cell Therapy Market Opportunities And Strategies

24. Stem Cell Therapy Market, Conclusions And Recommendations

25. Appendix

Companies Mentioned

For more information about this report visit https://www.researchandmarkets.com/r/3yzskj

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Stem Cell Therapy Global Market Report 2022: Rapid Growth in Emerging Markets & An Increase in Investments in Cell and Gene Therapies Driving...

According toGrand View Research, the global cell therapy market was valued at $7.8 billion in 2020 and is expected to expand at a compound annual growth rate (CAGR) of 14.5% between 2021 and 2028.

The rising number of clinical studies for cell-based therapies and investments in the industry may have a symbiotic relationship. The industry is seeing a snowballing number of ongoingclinical trialswith funding from governments and private agencies.

Theres an arguably thin line between cell and gene therapy. Cell therapy is the transfer of intact, live cells into a patient to help lessen or cure a disease, according to theAmerican Society of Gene and Cell Therapy (ASGCT). The cells may originate from the patient (autologous cells) or a donor (allogeneic cells).

Gene therapy involves the transfer of genetic material, usually in a carrier or vector, and the uptake of the gene into the appropriate cells of the body. Some protocols use both gene therapy and cell therapy.

Companies are using thebuilding blocks of lifeand advanced technologies to improve the treatment of human diseases and disorders such as cancer, providing an alternative to traditionally relied-on drugs and surgical treatments.

Cell therapy companies like Longeveron Inc. LGVN, Biogen Inc. BIIB, Alzamend Neuro Inc. ALZN and Solid Biosciences Inc. SLDB, as a result, have gained attention for their progress in using living cells to treat previously incurable diseases and disorders.

COVID-19 has reportedly causedsignificant disruptionto the cell and gene therapy industry. The pandemic has exacerbated the woes of an industry thats had its fair share of challenges with the supply of materials and the manufacturing and logistics processes.

General investments also slowed for the industry as governments shifted focus to saving lives and reviving economies. But things are starting to pick up now that the pandemic is on a downward trend.

Regulatory bodies like the Food and Drug Administration (FDA) have been urged to be more flexible in their approval timelines to make therapies affordable. Discussions continue around access and ensuring these therapies are affordable, reimbursable and profitable for the biopharmaceutical companies that develop them.

Academic and industry collaborations are expected to continue to expand and grow with noticeable impacts on the approval of products. Partnerships among academia, global pharmaceutical companies and small biotechs are expected to continue to shape the cell and gene therapy industry.

Longeveron, a clinical-stage biotechnology company, is one example of a company in the industry that has seemingly done well even during the pandemic. The company reports developing cellular therapies for investigation in chronic aging-related and certain life-threatening conditions.

The companys lead investigational product is Lomecel-B, a cell-based therapy product, derived from culture-expanded medicinal signaling cells sourced from the bone marrow of young, healthy adult donors.

Longeveron believes using the same cells that promote formation of new blood vessels, enhance cell survival and proliferation, inhibit cell death, and modulate immune system function may result in safe and effective therapies for some of the most difficult disorders associated with aging and some medical disorders.

Longeveron is sponsoring Phase 1 and 2 clinical trials in the following indications: Aging frailty, Alzheimers disease, metabolic syndrome, acute respiratory distress syndrome and hypoplastic left heart syndrome.

The companys mission is to advance Lomecel-B and other cell-based product candidates into pivotal Phase 3 trials to achieve regulatory approvals, subsequent commercialization and broad use by the healthcare community.

Photo by Edward Jenner from Pexels

This post contains sponsored advertising content. This content is for informational purposes only and is not intended to be investing advice.

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Is This Company In A Special Position Even As The COVID-19 Pandemic Affects Cell-Based Therapy Industry? - Benzinga

August 12, 2022 Ashish Shah, M.D., has assumed the newly created position of director of clinical trials and translational research and principal investigator in the Section of Virology and Immunotherapy at Sylvester Comprehensive Cancer Centers Brain Tumor Initiative (BTI) at the University of Miami Miller School of Medicine. Dr. Shah, who calls himself a quadruple Cane, returns to the site of his undergraduate studies, medical school, and residency as a faculty member. This follows a year-long fellowship at the National Institutes of Health, where he focused on clinical trial design and translational neuro-oncology.

Now, Dr. Shahs mission is to marry the clinical trials experience with his laboratory research and neurosurgery background to help the team bring novel therapeutics to patients with brain tumors.

We expect Dr. Shah to very quickly become one of the nations most recognized brain tumor researchers, said Ricardo J. Komotar, M.D., director of the Sylvester BTI and professor of neurological surgery at the Miller School. Dr. Shah is not only a world-class surgeon, but also does cutting-edge research. His role is to bring bench research to the clinic and bring clinical trials to our program.

This coupling of neurosurgery expertise with a dynamic research focus is rare, and Dr. Shah joins Michael Ivan, M.D., BTIs director of research, in fulfilling this dual role. Not only has Dr. Shah performed some of the most complex brain tumor surgeries, he has also published scores of papers on novel therapies and treatment approaches.

You need to make sure that the laboratory and the operating room for your patients are well connected, said Dr. Ivan. There are only a handful of programs in the country that build a bridge from bench to bedside in brain cancer research. It means developing a brain tumor treatment in the laboratory that can make it to a clinical trial and be translation-tested for rapid application to patients.

Most of Dr. Shahs work will focus on the highly aggressive glioblastoma type of brain tumor, which represents about half of all malignant brain tumors. Although nearly all glioblastoma tumors recur following removal, thanks to innovative approaches taken at the BTI, patients here have some of the best outcomes in the country.

I think the success of our world-class institute is largely due to a workflow weve implemented that allows us to maximize the amount of tumor we remove from the patients without compromising functionality, Dr. Komotar said. We do this through minimally invasive approaches like the laser thermal therapy, endoscopic approaches, fluorescence-guided surgery, and radiosurgery.

He points to the laser interstitial thermal therapy, an ablative procedure, as one of the most promising techniques perfected here. This approach enables tumor cell killing through a 2 mm incision, while inducing the immune system to attack the tumor. The technique is lengthening remission by years in some patients, and Dr. Shah looks toward leading clinical trials for this therapy in 2023 to extend its applications.

Much of Dr. Shahs research focus, however, will focus on viruses associated with brain tumors, which he sees as fundamental to understanding glioblastoma in particular, and which may underlie curative treatment that has been so elusive in this complex cancer.

Viral-based gene therapy uses viruses to deliver genes into cancer cells and, by changing their fundamental genome, make them more susceptible to cancer treatments. Working with colleagues, Dr. Shah recently discovered a key role of endogenous retroviruses in glioblastoma development, and is also working to develop virotherapy that involves delivering tumor-selective suicide genes, using a novel retrovirus.

This viral-based gene therapy approach reprograms cancer cells to be sensitive to harmless prodrugs, eliciting a robust anti-tumor immune response. In clinical trials, it has been shown to extend overall survival by several months for certain high-grade gliomas.

On the one hand, were trying to find out which viruses are causing the cancer, and on the other, were trying to use viruses to treat cancer, he said.

The team is planning future biomarker-driven virotherapy trials, as well as trials that will help predict which patients may benefit from certain therapies.

The treatments we have to date have failed. Now we are working to harness the immune system to recognize these tumors and fight them off. Potentiating the immune response against our brain tumors is critical, Dr. Shah said. If we can use retroviruses to both kill tumors and induce an immune response, that is where I think the future is.

Dr. Ivan, whose lab is adjacent to Dr. Shahs, is enthusiastic about the new collaboration. I think that we have developed a very comprehensive research program here, and were excited to move forward, he said. We really felt recruiting Dr. Shah to this new position offers our patients additional access to new treatment and, ultimately, better outcomes.

Dr. Komotar added, One of the most exciting parts of my job is seeing the growth of our brain tumor program over the last several years, not only in terms of the number and the volume of brain tumors that were taking care of, but also the world-class people we are surrounding ourselves with. Dr. Shah fits right into that. He was a top recruit this year.

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