e-Abstract Book - Symposium 2022

7th ANNUAL SYMPOSIUM ON CELL AND GENE THERAPY

(A Hybrid event)

1-3 September, 2022

Organized by:

CENTRE FOR STEM CELL RESEARCH
(a unit of inStem, Bengaluru)

Christian Medical College Campus
Bagayam, Vellore, India

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Dear Colleagues,
We take immense pleasure in welcoming you to the 7th Annual Symposium on Cell
and Gene Therapy. This platform continues to bring together basic scientists and
physicians from both academia and industry as well as all others interested in and
responsible for developing this field in the country. This field is advancing with
amazing speed with new transformational products coming to clinical care for various
unmet needs. Considerable progress has also been made in India with several clinical
trials initiated with CAR T cells and gene therapy for genetic disorders – including a
first in human study. Some of these advances could provide much needed cost-
effective solutions for several unmet health care needs in India. We therefore hope
that this meeting will provide the opportunity for us to understand these requirements
better and take steps to address them in the country.

The program this year will include topics and presentations which will represent the
advances in the field. These include applications of cell and gene therapy in
hemoglobin and, immune cell therapy, applications of iPSC technology, non-viral
nucleic acid transfer, industry updates and manufacturing along with regulatory
aspects as well as updates from the industry on some of the evolving technologies.

We are again fortunate to have among our speakers some of the global leaders in the
field. This meeting is always structured to facilitate discussion both during formal
presentations at the scientific sessions and through informal discussions and
interactions during the breaks for those present in person at the venue. We also hope
that we can follow-up on these deliberations after the meeting with suitable actions
to move this field forward in India.

We would like to thank all of you for joining us in this endeavour.
Team CSCR

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7TH ANNUAL SYMPOSIUM ON CELL AND GENE THERAPY

1st – 3rd September, 2022
PROGRAMME SCHEDULE

DAY-1: Thursday, 1st September, 2022

1:00 to 1:05 PM • Prayer by Chaplain
1:05 to 1:20 PM
1:20 to 1:25 PM • Welcome remarks: Dr. J V Peter, Director, CMC / Dr. Maneesha Inamdar, Director, inStem /
Dr. Solomon Satishkumar, Principal, CMC
India Time
1.30 to 2:00 PM • Closing remarks: Dr. Alok Srivastava, Head, CSCR
2:00 to 2:30 PM Session-1: APPLICATIONS OF iPSC TECHNOLOGY
Chair-Maneesha S. Inamdar
2:30 to 3:00 PM
Title Speaker Name
3:00 to 3:15 PM
3:15 to 3:45 PM Consistency and reliability of quality testing for induced Stephen Sullivan
Pluripotent Stem Cells Global Alliance for iPSC Therapies, UK

Assuring reproducibility: Sharing data on hPSC - lines and Andreas Kurtz
clinical trials Charité-Universitäts medizin Berlin and
Fraunhofer Institute for Biomedical Engineering,
Creation of a Haplobank- Initiative in India
Sulzbach, Germany
Break
Poster presentation Dolly Daniel
Christian Medical College and
Centre for Stem Cell Research, Vellore, India

Session-2: IMMUNE CELL THERAPY
Chair- Navin Khattry

India Time Title Speaker Name

3:45 to 4:15 PM CAR-T cell therapy in relapsed/ refractory DLBCL Hasmukh Jain
Advanced Centre for Treatment, Research and
4:15 to 4:45 PM CAR-T cell therapy for haematological malignancies or solid
tumours Education in Cancer, Mumbai, India

4:45 to 5:15 PM Donor derived anti-3rd party CD8 T Cells in haploidentical Avery D. Posey
stem cell transplantation and immune cell therapy University of Pennsylvania,

Philadelphia USA

Aloukick Kumar Singh
Centre for Stem Cell Research,

Vellore, India

5:15 to 5:30 PM Break

Session-3: GENE THERAPY
Chair-Soniya Nityanand

India Time Title Speaker Name

5:30 to 6:00 PM Nongenotoxic conditioning for transplant of genetically H. Trent Spencer
6:30 to 7:00 PM engineered HSCs: Is this the future for HSC-based gene Emory University School of Medicine,
therapy
Atlanta, USA
Gene Therapy for Hemophilia A - What are the current
options? Alok Srivastava
Christian Medical College and
6:00 to 6:30 PM Gene Therapy for Platelet Derived Factor VIII Production in Centre for Stem Cell Research, Vellore, India
Hemophilia A
David Wilcox
Medical College of Wisconsin,

WI, US

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

KEY NOTE ADDRESS
Chair- Alok Srivastava

7:00 to 8:00 PM Developing New Transformative Luigi Naldini
Approaches to Genetic Engineering of San Raffaele Telethon Institute for Gene Therapy
Hematopoiesis
and San Raffaele University, Milano, Italy

End of Day-1

DAY-2: Friday, 2nd SEPTEMBER, 2022

Session-4: TECHNOLOGY ADVANCES
Chair- Udaykumar Ranga

India Time Title Speaker Name
2:00 to 2:30 PM
Assessing and mitigating off-target effects in Toni Cathomen
2:30 to 3:00 PM therapeutic genome editing Institute for Transfusion Medicine and Gene Therapy,

Precise modelling and correction of a Germany
spectrum of β-thalassaemic mutations in
human erythroid cells by base editors Mohankumar Murugesan
Centre for Stem Cell Research, Vellore, India

3:00 to 3:30 PM IgG-cleaving endopeptidase enables in vivo Sebastien Lacroix-Desmazes
3:30 to 4:00 PM gene therapy in the presence of anti-AAV Centre de Recherche des Cordeliers, France
4:00 to 4:15 PM neutralizing antibodies
Arun Srivastava
Development of optimized (“Opt”) and University of Florida, Florida , USA
generation Y (“GenY”) AAVrh74 vectors for
gene therapy of muscular dystrophies Break

Session-5: INDUSTRY SYMPOSIUM
Chair- Cartikeya Reddy

India Time Title Speaker Name
4:15 to 4:45 PM
Cell therapy solutions for every step – from discovery to cure Prathap Naidu
4:45 to 5:15 PM Thermo Fisher Scientific,

5:15 to 6:00 PM Hyderabad, India

Speaker Time Droplet Digital PCR for Cell and Gene Therapy — ddPCR Ajay Deepak Baskaran
6:00 to 6:10 PM resolves problems with qPCR Bio-Rad Laboratories (India) Pvt Ltd.,

Haryana, India

Poster presentation

Session-6: MANUFACTURING AND REGULATORY ASPECTS IN CELL AND GENE THERAPY
Chair- Cartikeya Reddy

Title Speaker Name

Manufacturing of Lentiviral Vector (LV) for Haemophilia A William Swaney
Expression Manufacturing LLC, Georgia, USA

Lentiviral mediated genetic modification of HSC from Gurbind Singh
6:15 to 6:30 PM Haemophilia A patients Centre for Stem Cell Research, Vellore, India

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

6:30 to 7:00 PM Advanced HSC clinical trials in the Necker hospital GMP facility Elisa Magrin & Jihene Benlagha

Necker-Enfants Malades Hospital, Paris, France

7:00 to 7:30 PM Virus-specific T Cell Engineering for Clinical Applications Adrian Gee
Center for Cell & Gene Therapy,
KEY NOTE ADDRESS Baylor College of Medicine, Houston, TX, US
Chair-RV Shaji
Mark C. Walters
7:30 to 8:30 PM Evolution of lentiviral vector mediated gene modified autologous UCSF Benioff Children's Hospitals,
HSCs for the treatment of hemoglobin disorders
California, USA
End of Day-2

DAY-3: Saturday, 3rd SEPTEMBER, 2022

Session-7: CELL AND GENE THERAPY- INDUSTRY UPDATES
Chair- Vijay Chandru

India Time Title Speaker Name
2:00 to 2:30 PM
2:30 to 3:00 PM Future of Vaccinology-mRNA Ajay Singh
Gennova Biopharma Ltd.,
3:00 to 3:30 PM
3:30 to 3:45 PM Pune, India

India Time CAR-T cell Therapy-Bench to Bedside-the Immuneel approach Anil Kamat
3:45 to 4:15 PM Immuneel Therapeutics Private Limited,
4:15 to 4:45 PM
Bangalore, India
4:45 to 5:15 PM
5:15 to 6:00 PM Allogeneic, Off the Shelf, Pooled, Bone marrow derived Pawan Kumar Gupta
Mesenchymal Stromal Cells (Stempeucel®) – a Potential break Stempeutics Research Pvt. Ltd.,
through therapy for Grade II & III Osteoarthritis knee
Bangalore, India
Break

Session-8: GENE EDITING Speaker Name
Chair- Souvik Maiti
Title Justin S Antony
University of Tübingen, Germany
A Mutation-Agnostic Hematopoietic Stem Cell Gene editing approach
Annarita Miccio
Genome editing approaches for beta-hemoglobinopathies Institute of genetic diseases Imagine,
INSERM UMR1163, Universite’ Paris
The CD34+CD90+HSCs as a target cell for CCR5 gene editing
Cite’ Paris, France
Saravanabhavan Thangavel
Centre for Stem Cell Research,

Vellore, India

Poster presentation

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session-9: NON-VIRAL NUCLEIC ACID TRANSFER
Chair- Arabinda Chaudhuri

India Time Title Speaker Name
6:00 to 6:30 PM
6:30 to 7:00 PM Smart lipid nanoparticle enabled mRNA vaccine for SARS-CoV2 Srujan Marepally
7:00 to 7:30 PM Centre for Stem Cell Research,
Engineering mesenchymal stem cells by modulating tissue
factor gene expression using short interfering RNA Vellore, India

Oommen P. Oommen
Faculty of Medicine and Health Technology,

Tampere University, Finland,

Boosting intracellular delivery of mRNA therapeutics Gaurav Sahay
and its applications Department of Pharmaceutical Sciences,

End of Day-3 Oregon State University, Oregon, US

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Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

LUIGI NALDINI Plenary
San Raffaele Telethon Institute for Gene Therapy and San Lecture - 1
Raffaele University, Milano, Italy

Developing New Transformative Approaches to Genetic
Engineering of Hematopoiesis

Hematopoietic stem cells (HSC) gene therapy (GT) by lentiviral vectors is providing
substantial benefit to patients affected by primary immunodeficiencies,
hemoglobinopathies and storage disorders. Long-term follow up shows stable
hematopoietic reconstitution by high numbers of corrected HSC without signs of clonal
expansion or exhaustion, providing a reassuring molecular picture underlying the long-
lasting clinical benefit.
Precise engineering by gene editing may further improve the safety of HSC GT by
achieving in situ gene correction or targeted transgene integration. We reported the first
targeted gene editing of human HSC followed by studies highlighting barriers limiting its
efficacy and novel strategies overcoming them. Homology-driven repair, however,
remains limiting. We now report that the choice of template can increase efficiency and
safety of the procedure. Moreover, the emergence of base and prime editors that
minimize or bypass the requirement for DNA DSB allows editing single/few mutant
nucleotides with limited activation of DNA damage response.
Another long-sought goal of HSC GT is to make space for the infused cells without relying
on genotoxic conditioning, which entails acute and chronic serious adverse effects. We
report that HSC mobilization opens a window of opportunity for engraftment of donor cells,
which can effectively outcompete those in the circulation for engraftment in the depleted
niches. Competitive advantage results from the rescue in culture of a detrimental impact
of mobilizing agents on HSC and can be further enhanced by transient over-expression of
engraftment effectors. These findings were obtained in mouse models of diseases to prove
their therapeutic potential and in human hematochimeric mice to validate them for
human HSC.
Overall, our work should advance HSC GT by a combination of transformative approaches
leveraging on precision genetic engineering while alleviating the morbidity of the
procedure, broadening application to several diseases and patients worldwide.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

MARK C. WALTERS Plenary
UCSF Benioff Children's Hospitals, California, USA Lecture - 2

Evolution of lentiviral vector mediated gene modified autologous
HSCs for the treatment of hemoglobin disorders

The development of autologous gene therapy approaches in clinically significant
hemoglobin disorders has the potential to broaden opportunities for curative outcomes.
This advance hinges on favorable safety/efficacy profiles of the therapy in the short- and
long-term and on assuring expanded access to the novel therapy. Lentiviral transduction
of hematopoietic stem progenitor cells (HSPCs) in the current manufacturing era reliably
transduces 70% or more of the cells in recipients with transfusion-dependent β-thalassemia
(TDT) and sickle cell disease (SCD), which is sufficient for red blood cell transfusion
independence in ~90% of TDT recipients and resolution of acute vaso-occlusive episodes
(VOEs) in SCD, although ineffective erythropoiesis persists in both disorders. As of August
2021, 63 patients with TDT had received a gene therapy treatment sponsored by bluebird
bio, Inc with a median of 41.4 (9.0–87.5) months followup. Patients in phase 3 trials were
more likely to experience transfusion independence in phase 3 (34/38; 89.5%) than in
phase 1/2 (15/22; 68.2%) trials, illustrating improvements in manufacturing. In parallel, a
group of 35 patients with SCD who received the same lentiviral vector also had a
significant clinical benefit. The median total Hb level increased from 8.5 g/dL at baseline
to ≥11 g/dL from 6 months through last visit without RBC transfusions and the anti-sickling
HbAT87Q contributed at least 40% of total Hb. Of 29 evaluable patients, 28 had a complete
resolution of severe VOEs after infusion as compared with 3.5 events per yr (1.0–13.5) in the
2 yrs before enrollment.

Another approach of lentiviral therapy has targeted inactivation of BCL11a, the master
regulator of globin switching. The lentiviral vector in this study expresses a modified miRNA
under erythroid regulatory control that has a potent inhibitory post-transcriptional effect
on BCL11a. In a study conducted by D. Williams and colleagues at Boston Children’s
Hospital, the results of a phase I trial showed HbF levels in the 20 – 40% range, post-infusion.
This was accompanied by a significant clinical benefit in the majority of patients. Together,
both approaches of lentiviral gene therapy for hemoglobin disorders have shown a strong
clinical benefit associated with improved transduction of autologous HSPCs. Concerns
about long-term risks, including clonal hematopoiesis, will be presented.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session-1: APPLICATIONS OF iPSC TECHNOLOGY 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

Stephen Sullivan
Global Alliance for iPSC Therapies, UK

Consistency and reliability of quality testing for induced
Pluripotent Stem Cells

Introduction
The desire to generate tailored cells for replacement cell therapies has led to the current
focus on induced pluripotent stem cell (PSC) manufacture. Unlike cells of adult tissues,
iPSCs are both expandable in culture (self-renewal) and can be induced to differentiate
into all three hundred or cell types of the human body (pluripotent).
However, cultures of such cells can be heterogenous and unstable if not carefully
monitored. They exhibit differences in morphology and growth kinetics in response to
changes in culture conditions and passaging approaches. Many often rely on qualitative
observation to assess culture readiness with demonstrated variability across users. This has
significant consequences in manufacturing where scaling magnifies variability and makes
cultures hard to control. The resulting iPSC clones may vary from batch-to-batch or from
each other within the same batch, in their potential to differentiate into desired cell type,
affecting the process efficiency, robustness and predictability. Therefore, it is important
that clear standards and metrics are identified and adopted because these cells
represent the foundation for which PSC therapeutic development is vested.

Recommendations
Recommended actions are to: (i) adopt accurate, context specific and consistent use of
terminology when discussing the development and manufacture of iPSC therapies; (ii)
adopt a quality-by-design mindset for process development; (iii) be aware that early
decisions have significant and lasting consequences on Chemistry, Manufacturing and
Controls (CMC) and clinical impact and make sure such decisions are taken after their
consequences are understood (iv) engage other professional functions early to share best
practice and develop a complete translational roadmap for the final therapeutic product
you wish to manufacture; (v) identify critical quality attributes required for the starting
cellular materials, process intermediates, and final therapeutic; (vi) know the limitation of
your tools and employ orthogonal testing where necessary; and (vii) support efforts to
harmonize regulatory requirements for iPSC therapeutics globally.

Conclusion
Here we relay recommendations for manufacturers to ensure efficient translation of iPSC
therapeutics overall. We also detail useful training resources to assist with the skills shortages
currently experienced in this rapidly expanding sector.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session-1: APPLICATIONS OF iPSC TECHNOLOGY

Andreas Kurtz
Charité-Universitäts medizin Berlin and Fraunhofer Institute for
Biomedical Engineering, Sulzbach, Germany

Assuring reproducibility: Sharing data on hPSC - lines and clinical trials
Human pluripotent stem cells (hPSC) can be established from any person and have the
potency to differentiate into all cell types of the human body. Hence, hPSC are
predestined to generate biological avatars of persons to serve as disease and organ
models, or be used in cell therapy. The thousands of genetically unique hPSC - lines
established worldwide provide a huge resource for research and medicine. To fully explore
this resource, it is essential to make the data associated with each hPSC - line available in
an unambiguous, accessible, findable and re-usable manner. Furthermore, reproducibility
of results depends on a clear definition and identity of the material used to generate these
results. The means and needs to facilitate this data exchange will be outlined using the
example of the human pluripotent stem cell registry (hPScreg) and its features. The Registry
provides each cell line a unique identifier to which data can be globally annotated. Some
key data are registered directly in the database, while others are linked to other resources.
All data are publicly accessible. Importantly, the registry testifies to the ethical provenance
and scientific quality of each registered line by issuing certificates. The Registry is supported
by an international network of stakeholder organizations and key researchers with the goal
to serve the needs of the community and make data a key resource for international
research.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session-1: APPLICATIONS OF iPSC TECHNOLOGY 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

Dolly Daniel
Christian Medical College and Centre for Stem Cell Research
(a unit of inStem, Bengaluru), Vellore, India

Creation of a Haplobank- Initiative in India

The significant contribution of cellular therapeutics to regenerative medicine, took an
interesting and new approach following the development of induced pluripotent stem
cells (iPSCs) by Shinya Yamanaka and colleagues in 2006. Banking of iPSCs for this purpose
became a concept. However immune compatibility , particularly of HLA is critical, and
the cost and feasibility of developing personalized cell lines remains prohibitive.
Considering the extremely high polymorphism of the HLA locus, and the vast diversity
across different ethnic populations the concept of “haplobanking” emerged, where iPSCs
are derived from HLA homozygous individuals sharing haplotypes which will offer the
maximal population coverage.

The Indian Scenario is complex. With it’s extremely wide genetic diversity, it differs from the
more homogenous populations around the world. Simulations of the estimated number of
homozygous HLA lines required to provide HLA-A, HLA-B and HLA-DR compatible tissue in
various populations worldwide have been undertaken for UK, Japanese , Chinese and
North Americans , and all have shown that between 50-150 HLA homozygous donors
would provide HLA-compatible tissue for around 50 to 90 % of the respective populations.
However this is expected to be significantly higher in India, with the possibility of unique
haplotypes in many of our populations

The other challenge relates to performing HLA typing exclusively for this purpose which
makes the cost of such a project prohibitive and unfeasible. The idea of tapping a
resource such as a stem cell registry with HLA typed healthy donors emerged, and a
project took shape. From 2 previous publications the top 10 – 20 haplotypes were
identified, and working together with DATRI, a stem cell registry, donors homozygous for
these haplotypes were contacted, and after appropriate informed consent, had samples
drawn for subsequent testing and generation of iPSCs.

Increasing the clinical applicability of iPSCs requires that the “starting material” is
appropriate and uniform, and that standard protocols for iPSC generation are used to
ensure global applicability. The consenting and infectious disease testing protocols have
been modified to be in keeping with GAIT requirements. To date 15 Clinical grade iPSCs
have been generated in a GMP facility, and evaluated for sterility, pluripotency and
genomic stability, and have found to meet all quality requirements

Coordinating with a stem cell registry, and keeping in tune with GAIT guidelines, a bank of
15 clinical grade iPSCs , sharing the top 10 most frequent haplotypes in the Indian
population (as per available literature) have been generated. Expanding this pool and
being part of a global haplobanking initiative will impact greatly in making regenerative
therapy available in our country.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

HASMUKH JAIN
Tata Memorial Centre Advanced Centre for Treatment, Research and
Education in Cancer, Mumbai, India
CAR-T cell therapy in relapsed/ refractory DLBCL
CAR-T cell therapy is a breakthrough treatment for relapsed/refractory DLBCL and has
changed the treatment paradigm. From universally dismal outcomes in this setting, we
have a possibility of cure in at least 40% of the patients. The progress in this field has been
so rapid that in a span of 5 years, the treatment is already approved in second-line
therapy. I will present an overview of the data and also present our work.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session 2: IMMUNE CELL THERAPY 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

AVERY D. POSEY
University of Pennsylvania, Philadelphia USA

CAR-T cell therapy for haematological malignancies or solid tumours
Chimeric antigen receptor (CAR) technology allows T cells to be genetically engineered
with a novel immune receptor (as well as additional pro-inflammatory or effector
molecules) that redirect T cell specificity based on the targets of monoclonal antibodies.
This genetic engineering allows the manufacturing of billions of T cells specific for one
tumor-associated antigen.
Previously, we designed a CAR-T cell therapy that targets the glycopeptide epitope Tn-
MUC1 and demonstrated anti-tumor efficacy against a variety of tumor histotypes,
including T cell lymphoma, pancreatic cancer, breast cancer, prostate cancer, and other
cancer histotypes. Now, we extend the targeting of Tn-glycopeptides to an oncofetal form
of fibronectin, recognized by the antibody FDC-6. Tn-fibronectin-targeting CAR T cells
exhibit similar efficacy against PC3 prostate tumors as CAR T cells targeting cell surface
antigens; however, the mechanism of action of cytotoxicity appears to be different,
suggesting that T cells targeting extracellular antigens may represent a novel platform for
inducing anti-tumor efficacy. Additional approaches for improving CAR T cell activity
against tumors will be discussed, including the use of enzymatic activity, the generation of
immunomodulatory fusion proteins, and secretion of large molecules, such as cytokines
and antibodies.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session 2: IMMUNE CELL THERAPY

ALOUKICK KUMAR SINGH
Centre for Stem Cell Research (a unit of inStem, Bengaluru), Vellore, India

Donor derived anti-3rd party CD8 T cells in haploidentical- hematopoietic stem
cell transplantation and immune cell therapy

Donor derived T cells play an important role in rapid immune reconstitution and
engraftment of hematopoietic stem cells in allogeneic-hematopoietic stem cells (HSCc)
transplantation. However, they are known to cause graft-vs-host disease (GvHD). To
overcome this limitation, during the several years of extensive research we have
demonstrated that the use of donor derived anti-3rd party CD8 T cells can not only
overcome the problem of GvHD but can also induce the engraftment of HSCs by
selectively depleting only alloreactive T cells by the virtue of veto activity. In the allogeneic
conditions, when anti-3rd party CD8 T cells are targeted as non-self by alloreactive host T
cells; they can induce the killing of attacking cell by inducing Fas-Fal mediated apoptosis.
We have developed the approach for the generation of anti-3rd party CD8 T cells
depleted of any possible alloreactivity and also determined in a graft vs. host (GVH) mice
model that donor derived CD8 T cells can induce engraftment of T- cell depleted HSCs in
MHC disparate transplantations without the risk of GvHD and rejection. In a preclinical
sickle cell disease model we have demonstrated that donor derived anti-3rd party T cells
can induce a higher percentage of donor type chimerism without the incidence of GvHD,
which was sufficient to reverse the sickle cell disease status. Moreover, when we tested
antigen-specific CD8 T (Ovalbumin specific) cells in mice infused with B16-OVA-tdTomato
Melanoma, we observed marked reduction in tumor progression. Recently, we also
developed an approach for the generation of human antiviral- T cells depleted of
alloreactive contamination. The approach is now being tested in phase1/2 clinical trial in
MD Anderson Cancer Center, in the context of a safer non-myeloablative haploidentical
T cell depleted Hematopoietic Stem Cell Transplant in elderly patients with malignant
hematological diseases. So far, results are encouraging. Based on our mouse models, this
very mild protocol is also open for patients with Sickle disease and Thalassemia.
Furthermore, currently we are in a process of developing universal off-the-shelf anti-CD19
CAR-T cells by using antigen specific CD8 T cells.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session-3: GENE THERAPY 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

TRENT SPENCER
Emory University School of Medicine, Atlanta, USA

Nongenotoxic conditioning for transplant of genetically engineered HSCs: Is this
the future for HSC-based gene therapy

Genetic engineering of hematopoietic stem and progenitor cells (HSPC) using
recombinant viral and non viral vectors is a promising strategy toward a lifelong cure of
many diseases. However, the primary risks associated with this approach center on the
requirement for pre-transplantation conditioning necessary to make space for, and
provide immune suppression against the potentially immunogenic transgene product.
Traditional conditioning agents utilize genotoxic mechanisms of action, such as DNA
alkylation, that increase risk of sterility, infection, and developing secondary malignancies.
We have been developing several strategies that eliminate the issues associated with
genotoxic conditioning, including the use of antibody-based immunotoxins and ligand
based chimeric antigen receptors. For example, we and others have used a non-
genotoxic conditioning protocol using an immunotoxin targeting CD117 (c-kit) to achieve
endogenous hematopoietic stem cell depletion and a cocktail of monoclonal antibodies
to provide transient immune suppression against the transgene product. Our proof of
concept studies using a murine hemophilia A gene therapy model shows this strategy
provides high-level engraftment of hematopoietic stem cells genetically modified ex vivo
using recombinant lentiviral vector (LV) encoding a bioengineered high-expression factor
VIII variant. Factor VIII procoagulant activity levels were durably elevated into the normal
range and phenotypic correction achieved. Furthermore, no immunological rejection or
development of anti-FVIII immunity was observed. These preclinical data support clinical
translation of non-genotoxic antibody-based conditioning in HSPC LV gene therapy for
hemophilia A.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session-3: GENE THERAPY

DAVID A. WILCOX
Medical College of Wisconsin, WI, US

Gene Therapy for Platelet Derived Factor VIII Production in Hemophilia A

Objective:

Hemophilia A (HA) is an inherited bleeding disorder affecting ≈1:10,000 people due to
genetic defects on the X-chromosome causing a deficiency of coagulation factor VIII
(FVIII). HA is commonly treated with a continuous infusion of FVIII protein replacement
therapy. Thus, our goal is to develop a safe, efficient, and effective long-term treatment
to control bleeding in HA. Our strategy involves transplantation of autologous
hematopoietic stem cells (HSC) transduced with a lentiviral vector (LV) encoding platelet
derived FVIII (Pleightlet™). Ectopic FVIII production and storage within platelet α granules
allows storage in an immune privileged fashion and regulated secretion of platelet derived
FVIII to directly repair broken blood vessels.

Methods:

Previous pre clinical studies showed that LV-HSC driven by the (ITGA2B) megakaryocyte
specific gene promoter permits FVIII to traffic into platelet α granules leading to long term
improved hemostasis in murine and canine HA models (even in the presence of inhibitory
antibodies in mice and without eliciting an immune response to FVIII in mice and dogs).
New engineering runs were designed to test and validate the ability of GMP clinical grade
LV HSC to undergo a clinical transduction protocol (CTP) producing human
megakaryocytes that synthesize and store FVIII within α granules for regulated secretion of
FVIII from platelets to establish hemostasis in HA.

Summary:

Results from seven engineering runs of the CTP validate that ex vivo gene transfer with the
LV “Pleightlet™" into normal HSC achieved significant synthesis and storage of biologically
active FVIII:C in megakaryocytes in vitro in a safe and efficient manner that was directly
proportional to LV copy number/HSC. The genetically modified HSC product passed all
release criteria for cell viability, sterility, and absence of recombinant LV. Further analysis
showed that the Pleighlet™ LV FVIII replacement-gene integrated into cellular DNA as
expected. In summary, these results demonstrate that the CTP can manufacture a safe
and effective Pleightlet™ LV HSC product that is acceptable for infusion into HA.

Conclusion:

Our Phase 1 clinical trial is open for recruitment (ClinicalTrials.gov NCT03818763) and
enrollment underway with FDA IND approval that utilizes LV transduced autologous HSC
encoding FVIII in platelets for treatment of HA. This single-center study is in progress for
safety testing this treatment for severe HA with potential to improve hemostasis long term
HA.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session-3: GENE THERAPY 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

ALOK SRIVASTAVA
Centre for Stem Cell Research (a unit of inStem, Bengaluru) &
Department of Haematology, Christian Medical College, Vellore, India

Gene Therapy for Hemophilia A - What are the current options?

As a monogenic disorder with major phenotypic improvements in outcomes with even
minimal increase in expression of Factor 8/9 in the blood, haemophilia has been a focus
of gene therapy research for over two decades. Adeno associated virus (AAV) vector-
based gene therapy for haemophilia A and B have the major focus of current clinical trials.
Different AAV vectors are being evaluated for their safety and efficacy and are in different
phases of clinical trials. One product for gene therapy of F8 deficiency based on a AAV5
vector has recently received a favourable review from the European Medicinal Agency
and is awaiting a final decision on market authorization. Several other potential gene
therapy products using different AAV serotypes (AAV8, AAV3 and other variants) are in
clinical trials. While safety of AAV has been excellent so far in haemophilia gene therapy
clinical trials, there have been very large variations on the peak and duration of
expression. Furthermore, two major issues can affect eligibility and utility of this approach
and these products in many developing countries. The first is the presence of pre-existing
anti-AAV neutralizing antibodies which will neutralize the product before it can reach the
hepatocytes. Current serology data suggests that there is a rather high incidence of anti-
AAV antibodies in the community in India, China and South Africa. The other major
concern is the fact that these products need to be administered after patients have
attained close to the adult liver size thus excluding young children below 10-12 years of
age.

Given these limitations, there is need to develop other strategies. The first such option is the
lentiviral vector approach. Conceptually, the F8 transgene is transduced into the
haematopoietic stem cells (HSC) with promoters which determine the cell lineage in which
it will be expressed. Two such trials have been initiated. A group at the Medical College of
Wisconsin, USA is evaluating expression of F8 in megakaryocytes / platelets of haemophilia
patients with inhibitors. We have initiated a clinical trial at the Christian Medical College,
Vellore, India in collaboration with the Emory University, Atlanta, USA where we are
evaluating a lentiviral vector carrying a high expression F8 gene with a monocytic lineage
promoter. This approach requires collection of mobilized HSC, their anti-CD34 antibody
based purification and then ex-vivo transduction followed by assessment of their suitability
for gene therapy mainly based on the vector copy numbers being in the defined range.
If this product is qualified for therapy, based on achieving pre-defined criteria, then these
cells are administered through an autologous HSC transplantation. This approach is
therefore a lot more invasive but has the advantage ultimately allowing treatment of even
very young children without any immunological barriers. Like for all gene therapy
approaches at present, careful follow-up will be needed to evaluate its safety as well as
efficacy.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session-4: TECHNOLOGY ADVANCES

TONI CATHOMEN
Institute for Transfusion Medicine and Gene Therapy, Germany

Assessing and mitigating off-target effects in therapeutic genome editing
Targeted genome editing with programmable nucleases, such as CRISPR-Cas and TALENs,
has heralded a new era in gene therapy. On the other hand, designer nuclease-
associated genotoxicity, such as off-target activity and chromosomal translocations,
poses a risk that must be carefully evaluated before clinical application of any gene
editing product. We recently introduced CAST-Seq, a diagnostic assay that detects
genome editing associated chromosomal aberrations with high sensitivity in clinically
relevant cells. In my talk, I will summarize the current state of clinical application of genome
editing, present off-target effects we identified with CAST-Seq, and describe some
mitigation strategies to reduce off-target effects, including the applications of alternative
gene editing strategies that can combine efficient editing with reduced genotoxic side
effects.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session-4: TECHNOLOGY ADVANCES 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

MOHANKUMAR MURUGESAN
Centre for Stem Cell Research(a unit of inStem, Bengaluru), Vellore, India

Precise modelling and correction of a spectrum of β-thalassaemic mutations in
human erythroid cells by base editors

β-thalassemia and HbE result from mutations in the β-globin locus that impedes the

production of functional β-hemoglobin and represents one of the most common genetic
disorders worldwide. Recent advances in genome engineering have opened up new
therapeutic opportunities to directly correct these pathogenic mutations using base
editors that install transition mutations (A>G and C>T) in the target region with minimal
generation of indels. Herein, for the first time, we demonstrate the usage of base editor in
the correction of point mutations spanning multiple regions of the HBB gene, including
promoter, intron and exon. To this end, we have engineered human erythroid cells
harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+
HSPCs with desired mutations for the primary screening by base editors. We further
performed precise creation and correction of individual HBB point mutations in human
erythroid cells using base editors, which were effectively corrected in the HBB-
engineered erythroid model. Intriguingly, most bystander effects produced by the base
editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our
study provides the proof-of-concept for the precise, efficient and scarless creation and
correction of various pathogenic mutations at the coding and non-coding regions
of HBB gene in human erythroid cells using base editors and establishes a novel
therapeutic platform for the treatment of β-thalassemia/HbE patients. This study can be
further explored in correcting the other monogenic disorders caused due to single base
substitutions.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session-4: TECHNOLOGY ADVANCES

SEBASTIEN LACROIX-DESMAZES
Centre de Recherche des Cordeliers, France

IgG-cleaving endopeptidase enables in vivo gene therapy in the
presence of anti-AAV neutralizing antibodies

Neutralizing antibodies to adeno-associated virus (AAV) vectors are highly prevalent in
humans. They block liver transduction and vector readministration. They thus represent a
major limitation to in vivo gene therapy. Overcoming anti-AAV antibodies generally relies
on immunosuppression and is not efficient in removing pre-existing antibodies. As a result,
a large majority of the patients are excluded from gene therapy. Imlifidase (IdeS – IgG
degrading enzyme) is an endopeptidase that degrades human IgG. Its efficacy was
recently validated in transplant patients and autoimmune setups. Our work demonstrates
that IdeS eliminates anti-AAV antibodies in the context of gene therapy. IdeS efficiently
cleaved pooled human IgG (intravenous Ig) in vitro. In mice passively immunized with
human IgG, IdeS administration decreased neutralizing anti-AAV antibodies and enabled
efficient liver gene transfer. The approach was scaled up to nonhuman primates, a natural
host for wild-type AAV. IdeS treatment before AAV vector infusion was safe and resulted
in enhanced liver transduction, even in the setting of vector readministration. Our results
provide a potential solution to overcome pre-existing antibodies to AAV-based gene
therapy.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session-4: TECHNOLOGY ADVANCES 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

ARUN SRIVASTAVA
University of Florida College of Medicine, USA

Engineering Genetically Enhanced T cells for clinical applications

In a phase I/II clinical trial sponsored by Solid Biosciences using the first generation of AAV9
vectors, serious adverse events were reported. In a trial sponsored by Pfizer, also using the
first generation of AAV9 vectors, several serious adverse events, and the death of a
patient, were also reported. Sarepta Therapeutics reported the results of a phase I/II trial
using the first generation of AAVrh74 vectors, with vomitting as the only adverse event,
indicating that AAVrh74 vectors are safer. However, the trial failed to reach its clinical
endpoint even at a high dose of 2x1014 vgs/kg that was was used. We have previously
reported that capsid-modified next generation (“NextGen”) AAVrh74 vectors (Mol. Ther.,
29: 159-160, 2021) and genome-modified generation X (“GenX”) AAVrh74 vectors (Mol.
Ther., 29: 184-185, 2021) are significantly more efficient than their wild-type (WT)
counterpart. In the present studies, we combined the two modifications to generate
optimized (“OptX”) AAVrh74 vectors, and documented that the transduction efficiency of
OptX AAVrh74 vectors was significantly higher both in primary human skeletal muscle cells
in vitro, and in mouse gastrocnemius (GA) and tibialis anterior (TA) muscles in vivo.
Furthermore, since the naturally occurring AAV contains a single-stranded DNA genome,
and expresses viral genes poorly, because ssDNA is transcriptionally-inactive, transgene
expression levels from recombinant ssAAV vectors are also negatively impacted. We
previously observed that the distal 10-nucleotides (nts) in the AAV2 D-sequence share
partial homology to the consensus half-site of the glucocorticoid receptor-binding
element (GRE), and that the glucocorticoid receptor signaling pathway is activated
following AAV2 infection/AAV2 vector transduction (Mol. Ther., 24: S6, 2016). We evaluated
whether substitution of the distal 10-nts in the D-sequence with the authentic full-length
GRE would lead to increased transgene expression from AAVrh74 vectors, termed
generation Y (“GenY”) vectors. The extent of the transgene expression from GenY AAVrh74
vectors was indeed significantly higher both in primary human skeletal muscle cells in vitro
as well as in mouse skeletal muscles in vivo. These studies suggest that the combined use
of the capsid-modified NextGen+GenY (“OptY”) AAVrh74 vectors may further reduce the
need for high vector doses currently in use, which has significant implications in the
potential use of OptY AAVrh74 vectors in the safe and effective gene therapy of muscular
dystrophies in humans.

This research was supported by a sponsored research grant from Sarepta Therapeutics.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session-5: INDUSTRY SYMPOSIUM

PRATHAP NAIDU
Thermo Fisher Scientific, Hyderabad, India

Cell therapy solutions for every step – from discovery to cure
The use of chimeric antigen receptor (CAR) technology has contributed towards
significant advances in the treatment of certain types of cancer. This technology harnesses
the immune defenses (e.g., T cells) to specifically target a patient’s cancerous cells with
modified immune cells carrying a CAR “payload”. As with many new technologies, rapid
progress is being made that overcomes the barriers and hurdles associated with earlier
generations of the CAR T cell technology.
In my presentation will discuss some of the more recent improvements to the development
and manufacturing of CAR T cell therapies, including approaches to T cell isolation,
engineering steps to produce CAR T cells, and strategies for the expansion of engineered
cells for subsequent patient treatment. Presentation will also cover the overview Thermo
Fisher Scientific CGT workflow solutions including the CTS Rotea system and Xenon
electroporation system.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session-5: INDUSTRY SYMPOSIUM 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

Ajay Deepak Baskaran
Bio-Rad Laboratories (India) Pvt Ltd., Haryana, India

Droplet Digital PCR for Cell and Gene Therapy — ddPCR resolves
problems with qPCR

It can be challenging to use quantitative PCR (qPCR) to achieve absolute quantification
of nucleic acid sequences from patient samples for analytical development,
manufacturing, quality control, and lot release of cell and gene therapies. The quality of
qPCR assays can be impacted by PCR inhibitors and the reference standard, affecting
accuracy, sensitivity, and reproducibility. With qPCR, the difference between operators
and labs can be over 100x.
In contrast, Bio-Rad's Droplet Digital PCR (ddPCR) technology provides reproducible results
with absolute quantification, eliminating the need for standard curves. By partitioning PCR
reactions into droplets, ddPCR permits direct counting of target sequences, giving you
unparalleled sensitivity and accuracy while reducing variability.
Leverage ddPCR technology to overcome quantification challenges reported with
existing methods for a wide range of applications, including:

• Viral titer quantification- improve quality control methods by minimizing the
influence of PCR inhibitors

• Vector copy number determination -accurately calculate copy number by using
probe chemistry with required specificity

• Detection of residual host cell DNA- achieve absolute quantification without the use
of reference genes, enabling testing of more samples per plate

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session 6: MANUFACTURING AND REGULATORY ASPECTS IN CELL AND GENE THERAPY

WILLIAM SWANEY
Expression Manufacturing LLC, Georgia, USA

Manufacturing of Lentiviral Vector (LV) for Haemophilia A

Haemophilia A is an x-linked recessively inherited bleeding disorder resulting from deficient
or defective coagulation factor VIII (fVIII) present in the bloodstream. The global incidence
is < 1 out of every 5,000 live male births making it the most common severe bleeding
disorder. Disease severity correlates with the fVIII mutation and resulting plasma fVIII activity
levels. Less than 1% activity is defined as severe disease and presents as spontaneous
bleeding leading to rapid joint morbidity, pain, and risk of hemorrhagic death. Levels in
the rage of 1 – 5% represent moderate disease and 6 – 50% represent mild disease with
most bleeding occurring following trauma. Therefore, even small elevations in fVIII activity
levels result in significant clinical improvement for patients affected with severe disease.
Various approved therapies exist including fVIII protein replacement using either
recombinant or plasma derived fVIII, bypassing therapies including recombinant
activated factor VII, and fVIII mimetic therapy with a bispecific antibody. Additionally,
multiple gene therapies are in late-stage testing including RNA-based antithrombin
knockdown and AAV-based fVIII gene replacement. However, all of these therapies are
expensive, and none represent a lifelong cure. The only curative therapy is liver
transplantation, which is not feasible for most patients. An alternative curative approach
under clinical investigation is transplantation of LV mediated genetically modified
autologous haematopoietic stem and progenitor cells containing a functional fVIII
transgene. This talk will describe the manufacturing process used to make the LV used in
one of these studies, highlight recent changes to this platform which increases expression
and improves safety, as well as discuss recent experiences with moving the process into a
fixed bed bioreactor needed for scale up and future commercialization efforts.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session 6: MANUFACTURING AND REGULATORY ASPECTS IN CELL AND GENE THERAPY 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

GURBIND SINGH
Centre for Stem Cell Research (a unit of inStem, Bengaluru), Vellore, India

Lentiviral mediated genetic modification of HSC from Haemophilia A patients
Lentiviral vectors mediated gene addition in hematopoietic stem and progenitor cells
(HSPC) is an attractive gene therapy approach for treatment of haematological disorders.
This approach allows the long term expression by stably integrating the gene of interest
into the genome. There are a number of clinical trials undergoing by using this approach
for primary immunodeficiency and hemoglobinopathies. Many lentiviral based products
have been approved, most recently being the FDA approval of Zynteglo for the treatment
of beta thalassemia.
Continued access of gene therapy products requires process development that leads to
low production cost and consistent procedures for transduction and quality assessment of
the genetically modified cells. At the Centre for Stem Cell Research, Christian Medical
College, we are using lentiviral vector mediated gene addition approach for treatment
of severe haemophilia-A patients. In collaboration with Emory University, we have
established a robust manufacturing process for modification of HSPC by using lentiviral
vectors expressing FVIII in monocyte specific manner. For this, we performed the apheresis
on three severe haemophilia A patients and enriched the CD34 cells by using Miltenyi
cliniMACS system. Transduction was performed by using our established procedures. Post
transduction analysis, including vector copy number were assessed and data was
submitted to Indian regulatory agencies to obtain the approval for phase 1 clinical trial.
Recently, we obtained the phase 1 clinical trial approval from the central and state
regulatory agencies. We successfully manufactured the drug product (CD34 cells
modified with FVIII lentiviral vector) in our academic GMP facility for conducting the phase
1 clinical trial.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session 6: MANUFACTURING AND REGULATORY ASPECTS IN CELL AND GENE THERAPY

ELISA MAGRIN & JIHENE KLIBI BENLAGHA
Necker-Enfants Malades Hospital, Paris, France

Advanced HSC clinical trials in the Necker hospital GMP facility

The cell and gene therapy facility is part of the cell and gene therapy laboratory
(Laboratoire de Therapie Cellulaire et Génique, LTCG), headed by Professor Marina
Cavazzana. It is located in the Biotherapy Department of Necker Hospital in Paris and it
has been designed to implement the development of clinical trials involving cells and/or
genes. This is a mixed structure of INSERM (Institut national de la santé et de la recherche
médicale, French National Institute of Health and Medical Research) and APHP
(Assistance Publique–Hôpitaux de Paris, the university hospital operating in Paris and its
surroundings). It has been accredited by the French Competent Authority (ANSM) in 2015
and is fully operational for the manufacturing of advanced therapy medicinal products
(ATMP). It offers the capacity of performing cells and gene therapy trials based on
research projects developed within the Imagine Institute in Paris. Thus, this research unit
enables the translational research, with the preclinical part conducted at the Imagine
Institute and the conduct of gene therapy and cell therapy clinical trials in the Biotherapy
Department of the hospital.
From the 2000s, many gene therapy clinical trials have been conducted by the Professor
Cavazzana group, using the ex vivo genetically modified hematopoietic stem cells
strategy, to treat genetic immune and hematopoietic disorders. While the first transduction
procedures were realized in a L2 laboratory, with few quality controls especially for the
environment, regulatory requirements increased in the last years and the LTCG needed to
upgrade to assure the quality of the medical products according to the authorities
obligations.
Today, the cell and gene therapy facility is composed of two clean rooms located in a
controlled atmosphere environment and of an isolator. Several clinical trials are ongoing,
in collaboration with French, European and international hospital center and industries.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session 6: MANUFACTURING AND REGULATORY ASPECTS IN CELL AND GENE THERAPY 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

ADRIAN GEE
Baylor College of Medicine, Houston, TX, US
Virus-specific T Cell Engineering for Clinical Applications
Viral infections pose a great risk tor immunosuppressed individuals, such as patients who
have received allogeneic hematopoietic stem cells transplants. Treatment with
pharmaceuticals is expensive, not always successful and infections often recur after
therapy. The viruses involved frequently express antigens to which T cells can be targeted.
In this presentation we report on the sequential development by the Baylor College of
Medicine Center for Cell and Gene Therapy of T cell-based treatments directed against
viral antigens and their clinical use. We describe the evolution of treatments ranging from
autologous cells targeting a single viral antigen, to the use banks of allogeneic T cells
targeting multiple viruses. The evolution of GMP- compliant rapid manufacturing of virus-
directed T cells will also be covered. Our experience in developing this cell therapy
application has resulted in >90% complete responses in the recipients. The lessons learned
from this experience are likely to influence the evolution of other types of cellular therapies
and are currently being used to target tumor associated antigens for cancer treatment.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session 7: CELL AND GENE THERAPY- INDUSTRY UPDATES

AJAY SINGH
Gennova Biopharma Ltd., Pune, India

Future of Vaccinology-mRNA
Vaccines are important biological tools involving Live attenuated, Protein subunits, DNA
and mRNA; that help in preventing infection by introducing the antigenic piece of any
pathogen into the body, thereby allowing the triggering of immune response and
preparing the body to fight that infection. Focusing our attention to the most advance
and promising mRNA platform, they are indeed revolutionizing the future path for
treatments against cancer and other infectious diseases because of the immense
benefits, primarily the shorter and less time consuming developmental process followed
by the economic benefits. As the candidate has set a benchmark with its potential as a
prophylactic measure in the SARS-CoV-2 pandemic its assistance is sure to be of great
importance in other viral diseases such as Influenza, Ebola, Zika, Rabies, Respiratory
syncytial virus (RSV), Chikungunya, Human Immunodeficiency virus (HIV), Cytomegalovirus
(CMV), Malaria, Tuberculosis, Hepatitis B, Cystic fibrosis.

There were certain challenges of this technology that were faced initially starting from
mRNA degradation making it unstable as a vaccine, unable to enter into the cells and
also causing severe inflammation followed by its storage at constant sub-zero
temperatures creating problems for transportation. But all these hurdles were overcome
by employing unique and robust methods such as using synthetic mRNA and also
encapsulating it in a lipid-based material that would easily slip into the cells. The
lyophilisation technique gain attention wherein the mRNA now could be easily stored at
cold temperatures without the need for sub-zero and that did not impact the efficacy of
the vaccine. This is a disease agonist technology that has a lot advantages over the
traditional vaccines in the context that the genetic sequences of many viral antigens can
just be altered keeping the basic platform un-disturbed

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session 7: CELL AND GENE THERAPY- INDUSTRY UPDATES 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

ANIL KAMAT
Immuneel Therapeutics Private Limited, Bangalore, India

CAR-T cell Therapy-Bench to Bedside-the Immuneel approach

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of
hematological malignancies but remains an unmet need in developing countries. In India,
low insurance coverage (<10%) and high out-of-pocket costs results in delayed detection
and sub-optimal therapy with resultant poor outcome, quality of life and high mortality.
There was no active CAR-T trial in India until 2021.
Immuneel Therapeutics was founded in 2019 with a vision to bring global quality and
affordable cell and gene therapy (CGT) to India. This presentation will outline Immuneel’s
journey from bench to bedside to provide CAR-T cell therapy to patients in India, set up in
midst of covid pandemic.
To meet the challenges of resource-constrained environment, Immuneel set-up
affordable, locally sourced infrastructure, logistics and education programs. Collaboration
with global partner/s to bring clinic-tested CAR-T cell assets to India enhanced regulatory,
physician and patient confidence. To improve access, availability, and affordability; local
solutions for maintaining chain of identity, custody, and condition for ultracold chain
logistics and manpower upskilling were developed.
India’s first-in-industry 12,000 sq ft integrated facility, inaugurated in January 2021 in
Bengaluru, is uniquely housed one floor above a high-volume bone marrow transplant unit.
The in-house education program for all stakeholders helped address covid-induced global
travel restrictions for international training. Algorithms for toxicity management including
cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity
syndrome (ICANS) were developed based on international protocols with 24/7 access to
prophylactic medications.
Immuneel’s phase-2 CD19 CAR-T cell therapy trial (CTRI/2022/03/041162) with a clinic-
tested asset, varnimcabtagene autoleucel (IMN-003A) for B cell malignancies (IMAGINE
study) is currently recruiting.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session 7: CELL AND GENE THERAPY- INDUSTRY UPDATES

PAWAN KUMAR GUPTA
Stempeutics Research Pvt. Ltd., Bangalore, India

Allogeneic, Off the Shelf, Pooled, Bone marrow derived Mesenchymal Stromal
Cells (Stempeucel®) – a Potential break through therapy for Grade II & III
Osteoarthritis knee

Background:

Osteoarthritis (OA) is the most prevalent joint disease and a common cause of disability.
We had conducted a randomized, double blind, multi-centric, Phase 3 study
(CTRI/2018/09/015785) to assess the efficacy and safety of intra-articular administration of
stempeucel® in patients with OA of Knee

Methods:

146 patients having Grade II & III OA based were randomized in the study. 73 patients
each received either a single intra-articular injection of stempeucel® (25 million cells) or
Placebo followed by 20mg hyaluronan and were followed up for 12 months. The primary
end point was evaluation at one year follow up of WOMAC Composite Index score as
compared to the placebo arm. Secondary end points were: WOMAC sub-scores – pain,
stiffness & physical function, VAS & MRI assessment of the articular cartilage.

Results:

65 patients from stempeucel® and 68 patients from placebo arm completed 12 month
follow up. WOMAC composite index, showed significant improvements in cell arm as
compared to placebo (mean difference: -632.74; P<0.0001; percentage change: -44.3%).
Stempeucel® significantly improved WOMAC pain, stiffness, physical function and VAS
scores at 6 and 12 months (P<0.0001) in stempeucel® arm.T2 mapping shows that there is
no worsening of the deep cartilage in the medial femoral tibial compartment in
stempeucel® arm whereas in placebo arm there is significant and gradual worsening of
the cartilage. Cartilage volume increased by 34.07 units in stempeucel® arm. CTX – II in
urine showed decrease in levels (-7.79 pg/ml) in stempeucel® arm. Anti – inflammatory
marker IL-10 decreased by -0.228 pg/ml in placebo arm whereas increased by 0.051 pg/ml
in stempeucel® arm. 5 AEs were possibly / probably related to the study drug and were –
injection site swelling and pain.

Conclusion:
Based on the data, CDSCO granted manufacturing and marketing approval for Grade 2
& Grade 3 Osteoarthritis in non – obese patients with BMI <30Kg/m2.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session 8: GENE EDITING 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

JUSTIN SELVARAJ
University of Tübingen,Germany
A Mutation-Agnostic Hematopoietic Stem Cell Gene editing approach
Metachromatic leukodystrophy (MLD) is a rare genetic disorder caused by mutations in
the Arylsulfatase-A (ARSA) gene. The enzyme plays a key role in sulfatide metabolism in
brain cells, and its deficiency leads to neurodegeneration. The clinical manifestations of
MLD include stagnation and decline of motor and cognitive function, leading to
premature death with limited standard treatment options. Here, we describe a mutation-
agnostic hematopoietic stem and progenitor cell (HSPC) gene therapy using CRISPR-Cas9
and AAV6 repair template as a prospective treatment option for MLD. Our strategy
achieved efficient insertions and deletions (>87%) and a high level of gene integration
(>47%) at the ARSA locus in human bone marrow–derived HSPCs, with no detectable off-
target editing. As a proof of concept, we tested our mutation-agnostic therapy in HSPCs
derived from two MLD patients with distinct mutations and demonstrated restoration of
ARSA enzyme activity (>30-fold improvement) equivalent to healthy adults. In summary,
our investigation enabled a mutation-agnostic therapy for MLD patients with proven
efficacy and strong potential for clinical translation.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session 8: GENE EDITING

ANNARITA MICCIO
Institute of genetic diseases Imagine, INSERM UMR1163,
Universite’ Paris Cite’ Paris, France
Genome editing approaches for beta-hemoglobinopathies
Beta-hemoglobinopathies are caused by mutations affecting adult hemoglobin β-chain
production. The only curative treatment is allogenic hematopoietic stem/progenitor cells
(HSPCs) transplantation, an approach limited by compatible donor availability and
immunological complications. Therefore, transplantation of autologous, genetically
modified HSPCs is an attractive therapeutic option. However, current gene therapy
strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally
effective in all the patients and/or raise safety concerns. Base editing is a CRISPR/Cas9-
based genome editing technology that allows the introduction of point mutations in the
DNA without generating dangerous double strand breaks. Dr. Miccio will discuss base
editing strategies aiming either to correct the genetic defect or target disease modifiers in
patient cells and provide a safe and effective treatment for β-hemoglobinopathies.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session 8: GENE EDITING 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

SARAVANABHAVAN THANGAVEL
Centre for Stem Cell Research (a unit of inStem, Bengaluru), Vellore, India
The CD34+CD90+HSCs as a target cell for CCR5 gene editing
Hematopoietic stem and progenitor cells (HSPCs) transplantation could potentially treat
few infectious diseases as well. Transplantation of allogenic HSPCs with C-C chemokine
receptor type 5 (CCR5) D32 genotype generates HIV-1 resistant immune cells. CCR5 gene
edited autologous HSPCs can be a potential alternative to hematopoietic stem cell
transplantation (HSCT) from HLA-matched CCR5 null donor. In this talk, we will show how
using a subpopulation of HSPCs namely
CD34+CD90+HSCs for gene editing could mitigate some of the problems associated with
HSPC gene therapy. We will share on how the approach improved the generation of CCR5
null immune system.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session-9: NON-VIRAL NUCLEIC ACID TRANSFER

SRUJAN MAREPALLY
Centre for Stem Cell Research (a unit of inStem, Bengaluru), Vellore, India

SMART lipid nanoparticle enabled mRNA vaccine for SARS-CoV2
During COVID-19 pandemic, mRNA vaccines have emerged as novel class of vaccines
found to be efficient in producing immunogenic responses against the virus. The antigenic
mRNA is delivered by lipid nanoparticles (LNPs). The efficiency of vaccine is critically
determined by the efficiency of lipid nanoparticles system. Existing mRNA based vaccines
by Moderna therapeutics, Pfizer/BioNTech are sing their LNPs. However none of these LNPs
are specific to dendritic cells (DCs). DCs play major role in antigen presentation. Towards
developing an mRNA vaccine with long lasting T-cell responses, we designed and
developed our own mannose mimicking lipid nanoparticle system to target mannose
receptor on DCs. DC targeting SMART LNPs (shikimoylated mannose receptor targeting
nanoparticle system) showed superior delivery into dendritic cells present in the lymph
nodes compared to non-targeted liposomal system.
We also optimized 5’&3’ UTRs that found to be equally efficient as Moderna reported UTRs.
Further, the modification of me1Ψ-UTP (1-methylpseudouridine) in mRNA showed efficient
translation in dendritic cells (DCs) and more stable with SMART nanoparticles. We designed
and validated artificial trimeric spike S1 domain (T-S1, mRNA-773) in western blot and also
evaluated their expressions and interaction with hACE2 in vitro. The pre-clinical studies
revealed that SMART-T-S1 mRNA vaccine induced strong neutralizing antibodies against
SARS-CoV-2 virus in mice model system. Further, T cells studies showed SMART-T-S1 mRNA
vaccine developed long lived memory T cells against SARS-CoV-2 spike protein and it
infiltrated into lungs of SMART- T-S1 mRNA vaccine injected mice.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

Session-9: NON-VIRAL NUCLEIC ACID TRANSFER 7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

OOMMEN P. OOMMEN
Tampere University and BioMediTech Institute, Finland

Engineering mesenchymal stem cells by modulating tissue
factor gene expression using short interfering RNA

Stem cell therapies offer promising solutions to treat diseases or conditions for which few
therapeutic options exist. They have the potential to repair, restore, replace, and
regenerate tissues, and could possibly be used to treat many medical conditions and
diseases. Despite intense interest in harnessing the immunosuppressive and regenerative
potential of MSCs, the results from clinical trials have been disappointing and inconsistent
which has significantly dented the enthusiasm in the cell therapy field. This is believed to
be due to poor viability and function of the infused stem cells, as the transplanted cells
trigger thrombotic activity and are injured by the host innate immune system after
allotransplantation. To address this challenge, we have designed an siRNA delivery system
that effectively silence Tissue Factor (TF) gene expression, which we anticipated to be
responsible for the activation of coagulation cascade after stem cell transplantation. We
designed a novel pluronic-based micelles as an siRNA delivery system by covalently
conjugating siRNA targeting TF gene (or CD142) employing disulfide exchange reaction.
This nanocarrier displayed efficient gene silencing (72%) of the target gene without
eliciting any adverse toxicity. Gratifyingly, TF knockdown (TF-KD) of human bone marrow
derived MSCs (BMSCs) significantly suppressed the instant blood mediated inflammatory
reactions as evidenced by reduced platelet aggregation and thrombin-antithrombin
complex formation. To our surprise, effective silencing of TF also enhanced the
differentiation potential of BMSCs and displayed higher paracrine signaling as it exhibited
enhanced stimulation upon exposure to endotoxin. Furthermore, the soluble factors
produced by TF-KD BMSCs and untreated BMSCs competently suppressed the pro-
inflammatory cytokine when supplemented to pro-inflammatory M1 macrophages. Thus,
silencing of TF in BMSCs could significantly improve the inherent stem cell survivability issue
encountered upon transplantation with enhanced immunosuppressive properties that
may increase the safety in existing BMSCs based therapies

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022 Session-9: NON-VIRAL NUCLEIC ACID TRANSFER

GAURAV SAHAY
Oregon State University, Oregon, US

Boosting intracellular delivery of mRNA therapeutics and its applications
The field of nanomedicine is moving from an age of renaissance towards industrial
revolution. In part due to the transformational impact of lipid nanoparticle (LNP) enabled
mRNA vaccines against SARS-CoV2. Our lab has worked extensively onto understanding
LNP design, structure, and its impact on intracellular delivery of mRNA. Endosomal
sequestration of LNPs remains a formidable barrier to intracellular delivery. Structure-
activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl
phytosterols into LNPs (eLNPs) causes 200-fold improvement in gene transfection and the
length of alkyl tail, flexibility of sterol ring and polarity due to -OH group is required to
maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to
spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit
higher cellular uptake and retention, potentially leading to a steady release from the
endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity
of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape.
Based on these findings we designed next generation LNPs for deliver mRNA for
extrahepatic gene delivery and editing i.e., for the treatment of cystic fibrosis, retinal
degeneration, and COVID-19 therapeutics. I will also discuss our recent data on delivery
of LNPs delivered mRNA in non-human primate eye. Our findings emphasize the need for
greater insights into surface topology and structural properties of nanoparticles, and their
subcellular interactions. Next generation LNPs that enable tissue and cell-type specific
delivery of genes and genome editors can revolutionize modern medicine.

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022

Center for Stem Cell Research, Vellore would like to acknoeledge
the support from the following companies

Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India

7th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2022
Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India