Abstract Book

6th ANNUAL SYMPOSIUM ON CELL AND GENE THERAPY

1 - 3 September, 2021

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 6th Annual Symposium on Cell
and Gene Therapy. This platform aims to bring together basic scientists and
physicians along with all others interested in and responsible for developing this field
in the country. This field is advancing with amazing speed. Its potential for clinical
translation is immense. What is more significant is that 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 reflects some of the subjects which are of current interest in the
world as well in the country and where there is active work going on for some time or
is being evolved at different centres in the country. These include applications of cell
and gene therapy in hemoglobin and ocular disorders, immune cell therapy,
applications of iPSC technology, non-viral nucleic acid transfer and manufacturing
along with regulatory aspects as well as updates from the industry oon some of the
evolving technologies.
We are fortunate to have among our speakers some of the global leaders in the field.
This meeting has been structured to facilitate discussion both during formal
presentations at the scientific sessions and through informal discussions and
interactions during the breaks. 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

6TH ANNUAL SYMPOSIUM ON CELL AND GENE THERAPY

1st to 3rd September, 2021

PROGRAMME SCHEDULE

1:00 to 1:05PM DAY-1: THURSDAY, 01st SEPTEMBER, 2021
1:05 to 1:15PM
1:15 to 1:25PM • Prayer by Chaplain
• Welcome and introductory remarks: Dr. J. V. Peter, Director,CMC / Dr. Anna Pulimood, Principal, CMC /
India Time
1:30 to 2:30 PM Dr. Apurva Sarin, Director, inStem/ Dr. Alok Srivastava, Head, CSCR
• Remarks by Dr. Renu Swarup, Secretary, Department of Biotechnology, Ministry of Science and
India Time
2.30 to 3:00 PM Technology, Govt. of India
3:00 to 3:30 PM
3:30 to 4:00 PM KEY NOTE ADDRESS Speaker Name
4:00 to 4:15 PM Chair: Alok Srivastava Thierry Vanden Driessche
4:15 to 5:00 PM Title Vrije Universiteit Brussel (VUB) & KU Leuven,

India Time Gene therapy and gene editing for genetic diseases and Belgium
5:00 to 5:30 PM cancer: a personal journey
5:30 to 6:00 PM
Session-1: APPLICATIONS OF iPSC TECHNOLOGY
6:00 to 6:30 PM Chair: Raghu Padinjat
6:30 to 6:45PM
Title Speaker Name

iPSC based disease modelling of haematological diseases RV Shaji
The EBiSC iPSC bank for disease research Centre for Stem Cell Research,

Vellore, India
Rachel Steeg
Fraunhofer UK Research Limited
Scotland, UK

iPSC-based disease-modelling of Fanconi anaemia Grant Rowe
Boston Children's Hospital,
Break
Poster session Boston, USA
Session-2: TECHNOLOGY ADVANCES
Chair: Maneesha S Inamdar

Title Speaker Name
Genome editing for Sickle cell disease
Martin H. Steinberg
Turning Genes into Medicines: Lessons Learned in the Boston University School of Medicine,
Pursuit of Gene Therapy for Hemophilia
Preferential expansion of hematopoietic stem cells Boston, USA
enhances gene-modified cell frequency for gene therapy Katherine A. High
Asklepios BioPharmaceutical, Inc. (AskBio) North
Break
Carolina, USA
Saravanabhavan Thangavel
Centre for Stem Cell Research,

Vellore, India

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

India Time Session-3: GENE THERAPY Speaker Name
6:45 to 7:15 PM Chair: Mammen Chandy Arun Srivastava
Title University of Florida College of Medicine,
7:15 to 7:45 PM
Development of capsid- and genome-modified AAVrh74 Florida, USA
vectors for gene therapy of muscular dystrophies David Williams
Harvard Medical School,
Validation of BCL11A as a Therapeutic Target in Sickle
Cell Disease: Results from a First-in-Human Clinical Trial Boston, USA

Lentiviral vector-based gene therapy for Haemophilia A Alok Srivastava
7:45 to 8:15 PM Centre for Stem Cell Research,

Vellore, India

End of Day-1

DAY-2: Thursday, 2ndSEPTEMBER, 2021

Session-4: MANUFACTURING AND REGULATORY ASPECTS IN CELL AND GENE THERAPY

Chair: Cartikeya Reddy

India Time Title Speaker Name

3:00 to 3:30 PM Manufacturing and clinical application of mesenchymal Timothy O'brien
stromal cells for diabetic complications National University of Ireland, Galway, Ireland

Manufacturing the vector and CAR-T cells in India - The Rahul Purwar

3:30 to 4:00PM nuts and bolts Indian Institute of Technology Bombay, Mumbai, India

Engineering Genetically Enhanced T cells for clinical Bruce Levine
04:00 to 4:30PM applications University of Pennsylvania,

Philadelphia, USA

4:30 to 4:45PM Break

Session-5: CELL AND GENE THERAPY FOR OCULAR DISORDERS

Chair: Radhika Tandon

India Time Title Speaker Name
4:45 to 5:15 PM
Regenerative medicine by epithelia: increasing the Graziella Pellegrini
5:15 to 5:45 PM
complexity University of Modena and Reggio Emilia, Modena,

Italy

Gene augmentation strategies for corneal conditions Arkasubhra Ghosh

Narayana Nethralaya Foundation,

Bengaluru, India

5:45 to 6:15 PM Advances in iPSC-derived outer retina tissue mimetic: Ruchira Singh
6:15 to 7:00 PM Implications for translational approaches towards cell and University of Rochester,
gene-based therapy New York, USA

Poster presentation/ Industry Symposium

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

Session-6: IMMUNE CELL THERAPY
Chair: Vineeta Bal

India Time Title Speaker Name
7:00 to 7:30PM
NK cell therapy in the treatment of liquid and solid tumours Rizwan Romee
7:30 to 8:00PM Harvard Medical School,
8:00 to 8:30PM
8:30 to 9:00PM Boston, USA

Developing genetically-engineered, clinically-scalable, γδ Trent Spencer
T Cells for the treatment of solid tumours and Emory University, School of Medicine, Atlanta, USA
hematopoietic cancers of children

The first indigenous CAR-T cells in India - From bench to Rahul Purwar
clinic Indian Institute of Technology Bombay, Mumbai, India
Veto cells in haplo-transplantation and immune cell
therapy Yair Reisner
MD Anderson Cancer Centre,
End of Day-2
Houston, USA

DAY-3: Friday, 3rdSEPTEMBER, 2021

Session-7: CELL AND GENE THERAPY- INDUSTRY UPDATES
Chair: R.V. Shaji

India Time Title Speaker Name
3:00 to 3:30 PM
3:30 to 4:00PM Quality by Design: Reagents and Support for hPSC-Derived Cell Kimberly Snyder
and Gene Therapies STEMCELL Technologies,

Vancouver, Canada

Camille Lemey
Europe (Q&A)

Development of a scalable adeno-associated virus production Ann-Christin Magnusson
process by transient transfection in suspension cells Cytiva, Uppsala, Sweden

4:00 to 4:30PM Allogenic Cell and Gene therapy workflows with NK Cells Mohan C. Vemuri
Thermo Fisher Scientific
4:30 to 4:45 PM Break
Maryland, USA
India Time Session-8: GENE EDITING
4:45 to 5:15 PM Chair: Rakesh Mishra Speaker Name
Mohankumar Murugesan
Title Centre for Stem Cell Research, Vellore, India
Identification of novel HPFH-like mutations by CRISPR base
editing that elevates the expression of fetal hemoglobin

5:15 to 5:45 PM Genome editing of hematopoietic stem cells for therapeutic Pietro Genovese
5:45 to 6:15 PM applications Harvard Medical School
6:15 to 7:00 PM
Base editing of haematopoietic stem cells rescues sickle cell Boston, USA
disease in mice
Jonathan Yen
St. Jude Children’s Research Hospital,

Memphis, USA

Poster presentation and Industry Symposium

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: Krishna N Ganesh

India Time Title Speaker Name
7:00 to 7:30 PM
7:30 to 8:00 PM Nanoparticle-mediated gene therapy strategies for mitigating Avinash Bajaj
8:00 to 8:30 PM inflammatory bowel disease Regional Center for Biotechnology,
Co-delivery of NS1 and BMP2 mRNAs for bone regeneration
Self-transcribing and replicating RNA based vaccine for New Delhi, India
SARS-CoV-2
Chantal Pichon
University of Orléans,

Orléans, France

Priya Prakash Karmali
Arcturus Therapeutics,

San Diego, USA

End of Day-3

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

THIERRY VANDENDRIESSCHE
Vrije Universiteit Brussel and KU Leuven, Belgium

Gene therapy and gene editing for genetic diseases and cancer:
a personal journey

The regulatory approval of multiple gene therapy products for genetic diseases and cancer
will forever change the face of modern medicine. This offers new hopes for the patients
and their families that are blighted by these potential life-threatening diseases. Despite this
progress, there is still a need to further augment the efficacy and safety of gene therapy
vectors while minimizing the risk of untoward immune reactions. To further improve the
therapeutic window of gene therapy vectors, we have specifically engineered the
expression cassettes to increase expression of the gene of interest allowing the use of lower
and thus potentially safer vector doses for liver and muscle-targeted gene therapy.
Alternatively, we have tested new hyper-functional transgenes or combination gene
therapies to further improve the therapeutic efficacy. These concepts were validated in
preclinical hemophilia and muscular dystrophy models. To refine the specificity of gene
therapy, we have been exploring the use of CRISPR/Cas-mediated gene editing to excise
pathogenic alleles for the treatment of dominant genetic muscle disorders (DM1) in iPS-
derived cells or for targeted gene inactivation in vivo. Finally, we have exploited this
CRISPR/Cas technology to generate universal “off-the-shelf” CAR T cells for
immunotherapy of lymphoid malignancies. The continuous improvement of gene transfer
and gene editing technologies yields unprecedented opportunities to broadly impact on
the field at large.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021 Session-1: APPLICATIONS OF iPSC TECHNOLOGY

RV Shaji
Centre for Stem Cell Research, and Christian Medical College Vellore, India

JiPSC based disease modelling of haematological diseases
Induced pluripotent stem cells (i{PSCs) are currently being extensively used for disease
modelling and understanding the molecular basis of haematological diseases. We are
focused on applications of iPSCs in understanding the pathogenesis of inherited bone
marrow failure diseases, Fanconi anaemia, Diamond Blackfan anaemia and congenital
dyserythropoietic anaemia. We create mutant iPSCs in two ways: one by the generation of
iPSCs from patients with these diseases and the other by creating mutations in a normal
iPSC line using CRISPR-Cas9 gene-editing methods. We have successfully generated from
the patients with the diseases and created gene-edited cells for modelling CDA. These
iPSCs mimicked these diseases in culture after differentiating them to haematopoietic cells.
Our future research will be focused on studying the mechanisms underlying the
pathogenesis of these diseases.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

RACHEL STEEG
Fraunhofer UK Research Ltd., Scotland, UK

The EBiSC iPSC bank for disease research

The European Bank for induced Pluripotent Stem Cells (EBiSC) is a centralised non-profit
repository for the storage, banking, quality control and distribution of research-grade
human induced Pluripotent Stem Cells (iPSCs). EBiSC welcomes iPSC lines generated
internationally and since 2014 has safeguarded iPSCs covering >45 disease areas from >20
different organisations, making iPSCs available to users via the EBiSC Catalogue
(www.EBiSC.org). iPSC line depositors (commercial and non-profit research organisations)
support open science by sharing both the iPSCs themselves as well as extensive datasets
detailing the disease background of the original tissue sample, the background of iPSC
reprogramming and cell line characterisation data – all of which is shared to users. iPSC
lines are then standardised prior to distribution using core cell culture protocols and quality
control screening, with ongoing developments in screening the integrity of gene edited
lines. A robust ethical and legal framework enables managed access sharing of associated
genomic and clinical iPSC datasets. Currently in a second project phase supported by IMI2
and EFPIA, EBiSC2 is also developing novel scalable protocols for iPSC expansion,
differentiation and cryopreservation, to support users transitioning into high volume
applications whilst also ensuring accessibility for non-expert users. The inclusion of iPSC
tool lines in these protocol developments enables rapid generation of derived cell types,
which are consistent, reproducible and functionally mature. A key example is the
development of a fully humanised in vitro neuron-astrocyte co-culture system
whereby iPSC-derived astrocytes support neurons (generated by inducible and temporary
overexpression of SOX9 and NGN2 respectively) to display functional properties
comparable to the gold standard used in electrophysiology. Thus, EBiSC strives to
consolidate, streamline and simplify iPSC tools and resources to support researchers
worldwide with an overarching goal of accelerating biomedical research and improving
health.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

GRANT ROWE
Boston Children's Hospital, Cambridge, USA

iPSC-based disease-modelling of Fanconi anaemia

Patients with Fanconi anemia (FA) are at high risk of developing myeloid malignancy. This
typically occurs in the settin g of bone marrow failure, where somatic mutations drive
quiescent hematopoietic stem cell (HSC) clones to undergo ectopic self-renewal with
impaired differentiation as dysfunctional pre-leukemic stem cells, manifesting as
myelodysplastic syndrome (MDS). Additional somatic mutations result in further
dysregulation of self-renewal resulting in frank acute myeloid leukemia (AML), a significant
source of morbidity and mortality in FA. This process of clonal evolution is a difficult clinical
challenge in FA given the sensitivity of FA patients to genotoxic chemotherapy agents
required to treat MDS and AML. Therefore, improved understanding of the process of
clonal evolution of failing HSCs toward pre-leukemic and leukemic stem cells would benefit
patients with FA through the derivation of improved surveillance strategies to guide the
use of curative allogeneic HSC transplantation as well as the delineation of novel molecular
vulnerabilities in FA-associated MDS and AML. In this study, we used gene editing of
human induced pluripotent stem cells from FA patients to gain understanding of clonal
evolution in FA. We find that somatic mutations commonly occurring in MDS and AML
confer quiescent FA hematopoietic progenitors with aberrant self-renewal and that the
incorporation of an activating oncogene mutation can trigger leukemogenesis. Gene
expression analysis showed that FA MDS and AML cells express ectopic stem cell
transcriptional signatures and lose signatures of inflammation and terminal differentiation
typical of FA hematopoietic progenitors. Comparative functional analysis of human FA
progenitors, MDS-like cells, and leukemia cells revealed that cell cycle checkpoints
hyperactivated in progenitors were blunted in MDS and AML cells as a result of somatic
mutations, and that AML cells continue to show genomic instability and accumulation of
DNA damage despite ongoing proliferation. Overall, these findings provide new insight
into clonal evolution of FA hematopoietic progenitors to MDS and AML in a renewable
human system and provide a platform for therapeutic discovery in myeloid neoplasms
associated with FA.

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

Session-2: TECHNOLOGY ADVANCES 6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

MARTIN H. STEINBERG
Boston University School of Medicine, Boston, USA

Genome editing for Sickle cell disease

Sickle cell disease (SCD) is the most common of the β hemoglobinopathies, a group of diseases
caused by mutations in the β-globin gene (HBB). HBB glu7val, the sickle hemoglobin (HbS)
mutation, allows deoxygenated HbS to polymerize. Polymerization damages the sickle erythrocyte
leading to vasoocclusion and hemolytic anemia. Fetal hemoglobin (HbF), encoded by 2 genes
(HBG2/HBG1) in the HBB gene cluster, can prevent the polymerization of HbS. As exemplified by
compound heterozygotes for HbF and gene deletion hereditary persistence of HbF, HbF levels >10
pg. in >90% of sickle erythrocytes can prevent most complications of SCD.
Gene therapy for SCD has used autologous CD34+ progenitor cells to: 1) add a HBB engineered to
prevent HbS polymerization; 2) downregulate the activity of HBG2/HBG1 repressors or their effects
on globin gene switching; 3) correct the HbS mutation. Three trials of gene therapy have reported
results in more than a few patients. They have shown that HbF or a “HbF-like” HBB can decrease
HbS polymerization to the extent that hematologic parameters are nearly normalized while
symptoms of SCD are abrogated. By inducing levels of HbF, or a polymerization-inhibiting HbA, to
>40% of all hemoglobin, most erythrocytes are spared HbS polymer-mediated damage.
Lentiviral-mediated addition of normal HBB containing glutamine at position 87 (HbAT87Q).
inhibits polymerization of HbS. Six to 15 months after myeloablative conditioning—the current
standard for all forms of gene therapy—and following infusion of HbAT87Q modified CD34+ cells,
total hemoglobin was 10-16 g/dL, markers of hemolysis were nearly normalized and HbAT87Q was
>40%; patients became asymptomatic.
Two genes account for >90% of HBG2/HBG1 repression. In preclinical studies, in vitro knockout of
1 these repressors, BCL11A, in CD34+ cells was associated with ~70% HbF. In erythroid progenitors,
BCL11A activity is mediated by a specific enhancer. In a clinical trial where CRISPR/Cas9 was used
to disrupt the BCL11A erythroid enhancer, 6 to 21 months after infusion of modified CD34+ cells,
total hemoglobin was 11.0-13.5 g/dL, markers of hemolysis were nearly normalized, HbF was 42-
46% and F-cells >90%; patients became asymptomatic.
Post-transcriptional downregulation of BCL11A expression in CD34+ cells using a sh mRNA directed
to the erythroid enhancer, resulted after 6 to 24 months in total hemoglobin of 10.5-11.0 g/dL,
nearly normalized markers of hemolysis, HbF of 30-45% and F-cells 70-80%; patients became
asymptomatic.
Homology-directed repair of the HbS mutation and base or prime editing of HbF repressor binding
sites in the HBG2/HBG1 promotors are also being actively investigated. Gene therapy trials of SCD
and β thalassemia have so far been highly efficacious. To be an effective modality, gene-based
cellular therapeutics will need to: 1) develop non-myeloablative, non-genotoxic conditioning
regimens; 2) improve methods for stem cell collection; 3) devise means to deliver in vivo “editors”
to erythroid progenitors; 4) deal with issues of potential myelodysplasia or myeloid neoplasia and
the possible off-target effects of gene editing.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

KATHERINE A. HIGH
Therapeutics, Asklepios BioPharmaceutical, Inc. (AskBio) North Carolina, USA

Turning Genes into Medicines: Lessons Learned in the Pursuit of Gene Therapy for
Hemophilia

Trials of gene therapy for hemophilia began in the late 1990s; initial trials used a range of
vectors, including retroviral, adenoviral, and AAV, and were mostly conducted by academic
or small biotechnology company sponsors. A little over two decades later, most trials are
utilizing AAV vectors, and are being conducted by large pharmaceutical or large biotech
companies. For both hemophilia B and hemophilia A, multiple Phase 3 studies are now fully
enrolled or have concluded. The Phase 1 studies on which these are based, in the case of
hemophilia B, have shown durable expression of Factor IX at levels in the range of 30-50%.
Published reports of Phase 1 data for hemophilia A are fewer and in at least one case have
shown a phenomenon not previously described in AAV-mediated, liver-directed gene
therapy, of transgene levels that peaked 10-12 months after vector infusion then steadily
declined over the ensuing two-three year period. Key technical advances that have enabled
the current late-stage studies include the use of a high-specific activity transgene for Factor
IX, improved understanding of determinants of human immune responses to AAV vectors,
and advances in AAV manufacturing. This presentation will discuss safety and efficacy
results in current studies of investigational gene therapies for hemophilia, how these
compare to currently available therapeutic options for the disease, and issues that remain
to be addressed in the development of gene therapy for hemophilia. These issues will be
discussed in the larger context of advances in gene therapy and gene editing for single
gene disorders.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

SARAVANABHAVAN THANGAVEL
Centre for Stem Cell Research, Vellore, India

Preferential expansion of hematopoietic stem cells enhances gene-modified cell
frequency for gene therapy

CD34+CD133+CD90+ hematopoietic stem cells (HSCs) are responsible for long-term
multi-lineage hematopoiesis and the high frequency of gene-modified HSCs is crucial for
the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the
ex vivo culture and gene manipulation steps of HSPC graft preparation significantly reduce
the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the
manipulation. Here, we identified a combination of small molecules, that preferentially
expands CD34+CD133+CD90+ HSCs over other subpopulations of adult HSPCs in ex vivo
culture. The preferential expansion enriches the HSCs in ex vivo culture, enhances the
adhesion and results in a 6-fold increase in the long-term engraftment in NSG mice.
Further, the culture enriched HSCs are more responsive to gene modification by lentiviral
transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-
fold in vivo. The yield of gene-modified HSCs obtained by the culture enrichment is similar
to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study
addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by
developing and demonstrating a facile HSPC culture condition that increases the frequency
of gene-modified cells in vivo. This strategy will improve the outcome of HSPC gene
therapy and also simplify the gene manipulation process.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021 Session-3: GENE THERAPY

ARUN SRIVASTAVA
University of Florida College of Medicine, USA

Development of capsid- and genome-modified AAVrh74 vectors for gene therapy
of muscular dystrophies

Duchenne muscular dystrophy (DMD) leads to progressive muscle weakness and degeneration. A
lack of a protein, dystrophin, is the main cause of DMD. The gene coding for dystrophin is the
largest known gene in humans. More than 1,000 mutations in this gene have been identified. Since
there is currently no cure for DMD, which is often fatal, several gene therapy trials have been, or
are currently being performed using various AAV serotype vectors. A chimeric AAV capsid variant,
AAV2.5, composed of the AAV2 capsid with 5 mutations from the AAV1 capsid, was first used a
phase I clinical trial for DMD, and was found to be safe and well-tolerated, but no clinical efficacy
was achieved (Mol. Ther., 20: 443-455, 2012). In a phase I/II clinical trial sponsored by Solid
Biosciences using AAV9 vectors, adverse events like complement activation and thrombocytopenia
causing renal damage and cardiopulmonary insufficiency were reported. In a recent trial sponsored
by Pfizer, also using AAV9 vectors, several adverse events such as acute kidney injury involving
atypical hemolytic uremic syndrome and thrombocytopenia were also reported (Mol. Ther., 29: 464-
488, 2021). Sarepta Therapeutics recently reported the results of its phase I/II trial using AAVrh74
vectors. Although the adverse events were minimal, such as vomiting, and led to the expression of
a micro-dystrophin gene, the trial failed to meet its primary functional end point using the first
generation of AAVrh74 vectors (Nat. Rev. Drug Discov., 20: 91, 2021). Thus, it is clear that further
refinements in AAVrh74 vectors are warranted such that therapeutic levels of the dystrophin protein
can be achieved, preferably at reduced vector doses since it has become increasingly clear that the
host immune response correlates directly with the AAV vector dose. For example, whereas a dose
of up to 1x1014 vgs/kg of AAV8 vectors was safe, a dose of 3x1014 vgs/kg has been shown to be
associated with the death of 3 patients in a gene therapy trial of X-linked myotubular myopathy
(Hum. Gene Ther., 31: 787, 2020). Although a dose of 2x1014 vgs/kg of AAVrh74 vectors has been
shown to be well-tolerated in patients with DMD (JAMA Neurol., 77: 1122-1131, 2020), it would be
desirable to achieve clinical efficacy at a significantly lower vector dose. We have generated capsid-
modified and genome-modified AAVrh74 vectors which transduce primary human skeletal muscle
cells and mouse muscle tissues up to 10-fold more efficiently. Based on our previously published
studies with other AAV serotype vectors (Hum. Gene Ther. Meth., 27: 143-149, 2016), we anticipate
that the use of the capsid + genome-modified AAVrh74 vectors would lead to significantly higher
levels of transgene expression at further reduced doses. These studies suggest that the use of these
modified AAVrh74 vectors may lead to the potentially safe and effective gene therapy of human
muscular dystrophies at reduced doses, without the need for immune-suppression.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

DAVID WILLIAMS
Harvard Stem Cell Institute Bauer Building, Cambridge, USA

Validation of BCL11A as a Therapeutic Target in Sickle Cell Disease: Results from a
First-in-Human Clinical Trial

Sickle cell disease (SCD) is characterized by hemolytic anemia, pain, and progressive organ
damage. These manifestations are ameliorated by sufficiently high erythrocyte fetal
hemoglobin (HbF) comprised of alpha and gamma globins because a high level of HbF
mitigates sickle hemoglobin polymerization and erythrocyte sickling. BCL11A is a repressor
of gamma globin expression and HbF production in adult erythrocytes. Its downregulation
is a promising therapeutic strategy for induction of HbF. We enrolled patients with SCD in
a single-center, open-label, pilot trial. The investigational therapy involved infusion of
autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes
an shRNA targeting BCL11A mRNA embedded in a microRNA (shmiR) allowing erythroid
lineage-specific knockdown. Patients were assessed for primary endpoints of engraftment
and safety, as well as hematologic and clinical responses to treatment. Six patients have at
least 6 months follow-up after receiving BCH-BB694 gene therapy. Median follow-up was
18 months (range: 7-29). All patients engrafted and adverse events were consistent with
effects of the preparative chemotherapy. All fully evaluable patients achieved robust and
stable HbF induction (%HbF 21.6-40.0% at latest follow-up) with broadly distributed HbF
in red cells cells 58.9-93.6%) and HbF per F cell of 9.0-18.6 pg/cell. Clinical manifestations
of SCD were reduced or absent during the followup period.
This study validates BCL11A inhibition as an effective target for HbF induction and provides
preliminary evidence that shmiR-based gene knockdown offers a favorable risk/benefit
profile in SCD.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

ALOK SRIVASTAVA
Centre for Stem Cell Research, Vellore, India

Lentiviral vector-based gene therapy for Haemophilia A

Gene therapy for haemophilia A is aimed at providing sustained levels of FVIII in the blood
adequate for providing haemostasis. Based on pre-clinical studies carried out so far, most
of the current clinical trials are being done with the adeno-associated virus vector-based
gene transfer to the hepatocyte though different approaches have been used in the past.
Existence of pre-existing neutralizing anti-AAV antibodies and loss of transgene expression
are some of the major limitations in the field that need to be overcome. Lentiviral vector-
based approach for gene therapy for haemophilia A is being pursued for several years.
Luigi Naldini’s group at the San Raffaele Institute in Milan, Italy has been working on in-
vivo administration of phagocytosis shielded lentiviral vector with the FVIII transgene and
have recently reported success in the non-human primate model. A clinical trial has been
initiated at the University of Wisconsin with lentiviral vector-based platelet targeted
expression of FVIII. In collaboration with colleagues at the Emory University, Atlanta, USA,
we have developed a lentiviral vector-mediated haematopoietic stem cell (HSC) based gene
therapy for haemophilia A using a CD68 promoter in a sequence modified FVIII transgene
to enhance its expression in monocytic cells. Preclinical studies published earlier have
shown its efficacy in achieving normal FVIII levels in the haemophilic mouse model. Similar
experiments repeated with transduced human HSC transplanted in the NSG mouse showed
with the significant production of human FVIII in this less than ideal model for in-vivo
testing. (Hum Gene Ther 2018; 29: 1183-1201) Based on this pre-clinical data as well as the
lentiviral gene therapy vector related manufacturing data, this was approved for a phase 1
clinical trial in USA. However, in India, the regulators asked for transduction protocols to
be tested with haemophilic human HSCs. We therefore harvested mobilized peripheral
blood HSCs from three patients with severe haemophilia A and used them to test the
transduction protocol. We could demonstrate >95% viability of these transduced cells
along with vector copy number of 0.6 to 1.2/cell. With this data, we now have approval for
a phase 1 clinical trial for gene therapy for haemophilia A using autologous transduced
HSCs with a promoter directed at expression in monocytic cells.

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

Session-4: MANUFACTURING AND REGULATORY ASPECTS IN CELL AND GENE THERAPY 6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

TIMOTHY O'BRIEN
National University of Ireland, Galway, Ireland

Manufacturing and clinical application of mesenchymal stromal cells for diabetic
complications

The prevalence of diabetes mellitus is increasing globally and complications result in a
major clinical burden to patients and health care systems. While medical management to
prevent complications has merit, the majority of patients do not reach recommended
targets for risk factors such as glycemic control, lipids, blood pressure and smoking. Tight
glucose control can also be difficult to achieve in the absence of hypoglycemia. My
laboratory has been interested in the use of mesenchymal stromal cells (MSCs) for the
treatment of diabetic complications, both microvascular and macrovascular. This interest
stems from the fact that MSCs have angiogenic, immunomodulatory and anti-inflammatory
properties. We have built a GMP facility for the regulated manufacture of these cells and
we have generated preclinical data which has been used to gain approval for clinical trials
in the EU. The translational pathway to clinical trials in critical limb ischemia, diabetic foot
ulcers and diabetic nephropathy will be described. We have focused on GMP compliant
manufacture of MSCs and the following issues will be considered in the presentation: tissue
source, methods of culture, use of cell sorting, autologous versus allogeneic approaches,
use of bioreactors and xeno free media for culture.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

RAHUL PURWAR
Indian Institute of Technology Bombay, India

Manufacturing the vector and CAR-T cells in India –The nuts and bolts
CAR-T cell therapy has demonstrated remarkable success in long-term remission of
relapsed or refractory B-ALL. Recently two drugs (KymriahTM and YescartaTM) against
CD19 malignancies have been approved by FDA after phase I/II clinical trials. However,
there is a global disparity in availability of CAR T cell treatment. This “intent to cure”
therapeutic platform is not yet available in India at any price. Considering socioeconomic
conditions of patients in our country, the current CAR-T-cell therapy will be unaffordable
to majority of the patients due to high cost. To harness this technology and bringing it to
the clinic in India at affordable-cost, there is a clear need of developing indigenous CAR-T
cell technology platform. In this talk I will discuss about some of the work we have been
doing to develop anti-CD19 CAR T cells from discovery to preclinical. In addition, we scaled
up manufacturing platform as per the industry standard and validated assays for product
quality control in a cGMP facility approved by Indian regulatory agency. Very recently, we
initiated the Phase I pilot clinical trials for B-ALL (paediatric, PI: Dr. Gaurav Narula) and
DLBCL (adult, PI: Dr Hasmukh Jain) at Tata Memorial Hospital, Mumbai. In this talk I will
discuss my journey of indigenous CAR-T cells from bench to bedside.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

BRUCE LEVINE
Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA

Engineering Genetically Enhanced T cells for clinical applications
Since the 1990’s, we have conducted clinical trials of gene modified T cells. Gene editing
has created T cells resistant to HIV infection. Chimeric antigen receptor (CAR) T cells
targeting CD19 on B cells leukemias and lymphomas have induced durable complete
responses in patients who are relapsed or refractory to all other available treatments. New
designs for genetically modified T cells include switches and potency enhancements that
will be required for targeting solid tumors. In one such approach, multiplex gene editing
was accompanied by lentiviral transduction of a T Cell Receptor against the cancer antigen
NY-ESO-1. The first use of CRISPR in the US in humans demonstrated that multiplex human
genome engineering is safe and feasible. Translation of these technologies from research
bench to clinical application requires knowledge of the critical quality attributes of the
engineered cell product and acceptable limits. The road forward for wide patient access to
engineered cellular therapies depends not only on scientific progress in targeting, gene
modification and cellular manipulation methods, but also on meeting automation,
engineering, clinical site onboarding, and health policy challenges.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021 Session-5 :CELL AND GENE THERAPY FOR OCULAR DISORDERS

GRAZIELLA PELLEGRINI
University of Modena and Reggio Emilia, Italy

Regenerative medicine by epithelia: increasing the complexity
Gene therapy, cell therapy, and tissue engineering have the potential to revolutionize the
treatment of disease and injury. Attaining marketing authorization for such advanced
therapy medicinal products (ATMPs) requires a rigorous scientific evaluation by the
European Medicines Agency— authorization is only granted if the product can fulfil
stringent requirements for quality, safety, and efficacy. However, many ATMPs are being
provided to patients under alternative means, such as “hospital exemption” schemes.
Holoclar (ex vivo expanded autologous human corneal epithelial cells containing stem
cells), a novel treatment for eye burns, is one of the few ATMPs to have been granted
marketing authorization and is the first containing stem cells. This review highlights the
differences in standards between an authorized and unauthorized medicinal product, and
specifically discusses how the manufacture of Holoclar had to be updated to achieve
authorization. The result is that patients will have access to a therapy that is manufactured
to high commercial standards and is supported by robust clinical safety and efficacy data.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

ARKASUBHRA GHOSH
Narayana Netralaya Foundation, Bengaluru, India

Gene augmentation strategies for corneal conditions
The cornea is the essential outmost part of the eye, responsible for two-thirds of the
refractive power of the eye. It is commonly affected by various kinds of injuries and
infections that lead to loss of clarity and subsequently compromises vision. Additionally,
the cornea is also afflicted due to genetic and acquired conditions such as endothelial
dystrophies, keratoconus, etc. In addition, a small proportion of subjects undergoing
refractive correction are affected by post surgical haze, an adverse outcome. Corneal
opacities account for about 5% of the global blindness burden and one of the major
sources of preventable blindness. Although corneal transplantation is a successful
procedure, the very limited availability of donor tissue leads to several million blind
worldwide annually. Hence it is critical to develop alternative solutions for corneal
conditions. Cornea is an attractive target for gene therapy due to ease of delivery and
monitoring of treatment efficacy. Adeno associated viral vectors for treatment of corneal
scarring shows considerable reduction in haze in animal models when the TGF-beta
pathway is targeted using various kinds of genes. Since direct topical application is
possible, gene therapy using nanoparticles is also found efficient. Such treatment
modalities may now be ready for clinical translation.

PRIYADARSINI KUMAR

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

RUCHIRA SINGH
University of Rochester, NY, USA

Advances in iPSC-derived outer retina tissue mimetic(s): Implications for
translational approaches towards cell and gene-based therapy

The outer retina is comprised of the photoreceptor cells, retinal pigment epithelium (RPE)
cells, and the underlying fenestrated choriocapillaris. Myriad eye diseases manifest at the
photoreceptor-RPE or RPE-CC interface where dysfunction of individual cell layers (e.g.,
RPE) can have negative consequences for the survival and function of the other cell layers
(e.g., photoreceptor and choriocapillaris. For example, primary RPE and photoreceptor
dysfunction and/or an impaired photoreceptor-RPE interface leads to retinitis pigmentosa
(RP), the most common cause of inherited vision loss for people aged 20-60 years.
Similarly, primary RPE and/or choriocapillaris dysfunction is postulated to cause
photoreceptor cell death and subsequent vision loss in age-related macular degeneration
(AMD), a blinding disease that affects people > 50 years of age and is estimated to impact
~288 million people worldwide by 2040. Notably, in pre-clinical models of retinal
degenerative diseases and early-stage clinical trials on human patients with AMD, stem-
cell derived retinal cells, including RPE and photoreceptor precursors have yielded
promising results. Similarly, iPSC-derived retinal cells have been used as a preclinical
model for AAV-mediated gene therapy. Although these results are promising, the current
approaches to cell-based therapy and as a pre-clinical model for gene therapy lack the
consideration for the multi-tissue pathology of diseases like AMD that would benefit from
targeting more than one cell layer (e.g both RPE and choriocapillaris for AMD) especially
in view of upholding the therapeutic benefit for extended period of time. Towards that
goal, in the current presentation, I will highlight the recent work from our laboratory that
has shown the feasibility of developing multi-layered retina tissue mimetics. Specifically,
I will talk about the development of physiologically relevant tissue mimetic of the outer
blood retinal barrier, the RPE-choriocapillaris complex, that apart from being relevant for
disease modeling and drug screening applications, has the potential to transform cell and
gene-based therapies for AMD and related maculopathie

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

Session-6 : IMMUNE CELL THERAPY 6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

RIZWAN ROMEE
Harvard Medical School, Boston, USA

NK cell therapy in the treatment of liquid and solid tumors

Natural killer (NK) cells hold great promise in cancer immunotherapy. Recent studies
demonstrate that cytokine induced memory-like (CIML) NK cells exhibit anti-tumor activity
in patients with acute myeloid leukemia (AML). However, AML in many cases is not fully
recognized by native NK cell receptors. We hypothesized that arming NK cells with an AML-
specific chimeric antigen receptor (CAR) would improve their anti-tumor responses. To
address this, CIML NK cells from peripheral blood were transduced via an unconventional
baboon-pseudotyped lentivirus carrying single-chain variable fragment (ScFv) sequence
that specifically recognizes a neoantigen derived from NPM1 mutation, which occurs in
30% of AML. By achieving 40-90% CAR editing, CIML NK cells display boosted cytokine
production and specific cytotoxicity against NPM1-mutated AML line and patient-derived-
xenograft targets. CAR NK cells-controlled AML burden in xenograft models, and enhanced
efficacy was achieved by integrating membrane-bound IL-15 in the CAR design to support
long-term persistence of CIML NK cells in vivo. Thus, efficient editing of optimized CAR
construct on CIML NK cells substantially improve their anti-tumor responses, shedding light
on utilizing CIML NK cells as a CAR platform for manufacturing “off-the-shelf”
immunotherapies against a variety of liquid and solid tumors in clinic. Using CIML NK Cell
CAR platform we are currently also testing them against mesothelin expressing solid
tumors including ovarian and pancreatic cancers. Anti mesothlin CIML CARs show
promising activity against mesothelin expressing cell lines as well as against patient derived
organoids tumors (PDOTs) without significant activity against mesothelin expressing
healthy cells. We hope to take NMP NK CARs and Mesothelin NK CARs to clinic in near
future.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

TRENT SPENCER
Emory University School of Medicine, Atlanta, USA

Developing genetically-engineered, clinically-scalable, γδ T Cells for the treatment
of solid tumours and hematopoietic cancers of children

Although chimeric antigen receptor technologies are among the most exciting advances
as anti-cancer therapeutics, the use of alpha beta T cells may not be useful against for many
targets or cancers. We have developed an optimized GMP compliant ex vivo expansion
process for gamma delta T cells, which is being used in a clinical trial for childhood
neuroblastoma. Although non-modified allogenic cells are being used for this trial, we have
also optimized technologies for the genetic engineering of these cells. This presentation
will focus on the ex vivo expansion and genetic engineering of gamma delta T cells.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

RAHUL PURWAR
Indian Institute of Technology Bombay, Mumbai, India

The first indigenous CAR-T cells in India - From bench to clinic
CAR-T cell therapy has demonstrated remarkable success in long-term remission of
relapsed or refractory B-ALL. Recently two drugs (KymriahTM and YescartaTM) against
CD19 malignancies have been approved by FDA after phase I/II clinical trials. However,
there is a global disparity in availability of CAR T cell treatment. This “intent to cure”
therapeutic platform is not yet available in India at any price. Considering socioeconomic
conditions of patients in our country, the current CAR-T-cell therapy will be unaffordable
to majority of the patients due to high cost. To harness this technology and bringing it to
the clinic in India at affordable-cost, there is a clear need of developing indigenous CAR-T
cell technology platform. In this talk I will discuss about some of the work we have been
doing to develop anti-CD19 CAR T cells from discovery to preclinical. In addition, we scaled
up manufacturing platform as per the industry standard and validated assays for product
quality control in a cGMP facility approved by Indian regulatory agency. Very recently, we
initiated the Phase I pilot clinical trials for B-ALL and DLBCL at Tata Memorial Hospital,
Mumbai. In this talk I will discuss my journey of indigenous CAR-T cells from bench to
bedside.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

YAIR REISNER
MD Anderson Cancer Centre, TX, USA

Veto cells in haplo-transplantation and immune cell therapy
We have demonstrated during the past 2 decades in different preclinical mouse models
that Veto Cells, by virtue of their “veto activity” can effectively promote immune tolerance
in T cell depleted bone marrow allografting under mild immune suppression , as well as in
cancer cell therapy using genetically modified T cells. Veto cells, when targeted as non-self
by anti-donor T cells in the recipient, can “veto” the killing signal and delete the attacking
anti-donor cognate T cells through an apoptosis-based Fas-FasL mechanism. Recently we
were able to generate human central memory CD8 veto T cells which are depleted of
potential GVH reactivity, and these cells are now tested in a 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 , the potential of VETO-CAR T cells, namely VETO cells transduced with CD19
CAR , as “off-the shelf” allogeneic Veto CAR-T cells , currently successful in preclinical
mouse models , will be tested in the near future in relapsing patients with CD19+ malignant
hematological diseases. Finally , considering that durable immune tolerance towards donor
MHC is induced by this veto-based haploidentical HSCT protocol , we envision using it as
a platform for tolerance induction in kidney recipients , so as to cease completely their
chronic treatment with harmful immune suppression agents.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021 Session-7: CELL AND GENE THERAPY- INDUSTRY UPDATES

KIMBERLY SNYDER
Stem Cell Technologies, Vancouver, Canada

Quality by Design: Reagents and Support for hPSC-Derived Cell and
Gene Therapies

The number of human pluripotent stem cell (hPSC) derived therapies moving towards the
clinic increases every year and STEMCELL is committed to supporting researchers along the
full path from discovery through to clinical application. We have developed a
comprehensive portfolio of cGMP manufactured products for the expansion of high quality
human pluripotent stem cells (hPSC) and describe how these validated products are
integrated in a complete hPSC-based workflow to ensure reproducibility. To further
address the needs of the field, a novel animal origin-free (AOF) hPSC maintenance medium,
TeSR™-AOF, made with animal-free raw materials to the secondary level of manufacturing
will be presented. TeSR™-AOF was designed with quality and safety in mind and formulated
to robustly support optimized cell quality, improved performance and reproducibility
across all cell lines. By designing products with quality and regulatory considerations at the
forefront we will continue to support research from discovery through to the clinic.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

ANN-CHRISTIN MAGNUSSON
Cytiva, Uppsala, Sweden

Development of a scalable adeno-associated virus production process by transient
transfection in suspension cells

Viral vectors have become increasingly used as means of gene transfer for specific tissue
or cell type modifications. Several viruses have been investigated for their use in cell and
gene therapy with Adeno Associated Virus (AAV) as the main vector for gene therapies. In
this study, an efficient and scalable cell culture process for AAV production was developed
by evaluation and optimization of each process step.
In this study, HEK 293T cells were successfully adapted to suspension culture in a serum-
free medium without any animal derived components. Triple plasmid transfection using
polyethylenimine (PEI) was optimized for different parameters and conditions using a
Design of Experiments (DoE) strategy in shake flasks. The optimal concentrations and ratio
of plasmids, PEI, temperature, transfection volume and incubation times were evaluated for
transfection efficiency and virus productivity. The conditions were further developed for
production in single-use bioreactor systems.
A qPCR assay was used for viral genome titer. For total virus capsid titer, a commercial
ELISA was used for quantification. In parallel a Biacore-based method was developed for
total virus capsid titer quantification. The percentage of full capsids was calculated as the
ratio between viral genomes and viral capsids. A transduction assay based on flow
cytometry was used to evaluate the infectious virus titer.
An optimized transfection protocol in shake flasks was generated based on the DoE studies
for AAV2, with titers above 1014 viral capsids/L. The protocol has also been evaluated with
other serotypes, such as AAV5, AAV8 and AAV9 and was confirmed to generate similar
productivities. A stirred tank single-use bioreactor process was designed and optimized
based on the shake flask results. Furthermore, a second process was developed in WAVE
bioreactors and gave similar productivities. Thus, scalable, robust and reproducible
production of AAV was developed from small scale shake flask production up to 20L in
single-use bioreactor systems.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

Mohan C. Vemuri
Thermo Fisher Scientific, Maryland, USA

Allogenic Cell and Gene therapy workflows with NK Cells
Natural killer (NK) cells have emerged as a promising therapeutic for solid and
hematological tumors. NK cells are a part of the innate immune system and respond to
anything they perceive as “non-self”, including malignant cells. Unlike T-cells, NK cells can
provide an anticancer response in an antigen-independent manner, allowing NK cells to be
a potential “off the shelf” allogeneic therapeutic product. They have the potential to be
safer, less expensive, and more effective than current engineered T-cell therapies.
Irradiated and genetically modified feeder cells are typically used to expand NK cells to
clinically relevant numbers. However, despite being lethally irradiated before use, the
feeder cells do pose safety concerns. Generating the number of NK cells required for
therapeutic use, without the use of feeder cells, continues to be a major challenge.
CTS™ NK-Xpander™ expands functional primary human NK cells to clinically relevant levels,
without the use of feeder cells. NK cell expansion does vary by a donor, however qualified
donors have shown up to 2000 fold expansion within 2 to 3 weeks. The expanded NK cells
are functional and able to kill multiple cancer cell lines of solid tumors after co-incubation.
CTS™ NK-Xpander™ allows translational researchers to generate a large number of
functionally viable NK cells for cell and gene therapies to run phase I and II clinical trials;
ultimately improving the quality of life of cancer patients.

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

Session-8:GENE EDITING 6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

MOHANKUMAR MURUGESAN
Centre for Stem Cell Research, Vellore, India

Identification of novel HPFH-like mutations by CRISPR base editing that elevates
the expression of fetal hemoglobin

Switching hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin
is an effective strategy for the treatment of beta-hemoglobinopathies. Fetal hemoglobin
expression is down-regulated in the postnatal period due to the interplay of transcription
regulators with the HBG promoters. However, in the hereditary persistence of fetal
hemoglobin (HPFH) condition, naturally occurring point mutations in the HBG promoter
causes continued expression of fetal globin even during adulthood. Inspired by this natural
phenomenon, we screened the proximal promoter of human HBG genes using adenine and
cytosine base editors to identify other nucleotide substitutions that could potentially lead
to elevated levels of fetal globin. Both the base editors efficiently and precisely edited at
the target sites with a minimal generation of indels and no deletion of one of the duplicated
HBG genes. Through systematic tiling across the HBG proximal promoter, we identified
multiple novel target sites that resulted in a significant increase in fetal globin levels.
Further, we individually validated the top eight potential target sites from both the base
editors and observed robust elevation in the fetal globin levels up to 47 %, without any
detrimental effects on erythroid differentiation. We further demonstrated that the
introduction of -123T>C and -124T>C HPFH-like mutations drives gamma-globin
expression by creating a de novo binding site for the master erythroid regulator KLF1.
Overall, our findings shed light on so far unknown regulatory elements within the HBG
promoter that normally mediates fetal globin silencing and identify additional targets for
therapeutic upregulation of fetal hemoglobin.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

PIETRO GENOVESE
Harvard Medical School, Boston, USA

Genome editing of hematopoietic stem cells for therapeutic applications

Gene editing in Hematopoietic Stem/Progenitor cells (HSPC) holds the promise to provide
a safe and effective therapeutic option for several diseases. Yet, despite its tremendous
therapeutic potential and the continuous advances in perfecting gene editing platforms,
editing by homologous direct repair (HDR) remains constrained in the primitive HSPC
subset by quiescence and low expression of the DNA repair machinery. Recently, we
applied a barcoding strategy to clonal tracking of edited cells and showed that editing
activates p53, which significantly shrinks the size and clonal repertoire of the human
hematopoietic graft in hematochimeric mice, although individual engrafted edited HSC
preserved multilineage and self-renewing capacity. Transient p53 inhibition enhanced graft
size and restored its polyclonal composition. We also succeeded in increasing HDR
efficiency in HSC by forcing cell cycle progression and upregulating components of the
HDR machinery through transient expression of the Adenovirus 5 E4orf6/7 protein, which
recruits the cell cycle controller E2F on its target genes. Our enhanced protocol allows HDR
editing efficiencies of up to 50% long-term HSC, without perturbing their repopulation and
self-renewal properties, thus paving its way to clinical translation. Thus, we selected the X-
linked hyper-IgM syndrome (HIGM1) as paradigmatic disease for which precise gene
editing can immediately offer measurable clinical advantages. HIGM1 is caused by
mutations of CD40LG, whose absence in CD4 T-cells impairs their helper signaling for B-
cell activation/immunoglobulin class-switching. Since its unregulated expression caused
lymphoproliferation/lymphomas, we designed a one-size-fit-all correction strategy to fix
CD40LG mutations while preserving its physiologic regulation. By exploiting a suitable
mouse model and patient derived cells, we confirmed the therapeutic potential of both T-
cell and HSPC therapies, providing the rational for a first in human clinical testing.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

Jonathan Yen
St. Jude Children’s Research Hospital, TN, USA

Base editing of haematopoietic stem cells rescues sickle cell disease in mice

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB1. We used a
custom adenine base editor (ABE8e-NRCH) to convert the SCD allele (HBBS) into Makassar
β-globin (HBBG), a non-pathogenic variant. Ex vivo delivery of mRNA encoding the base
editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs)
from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after
transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG
was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased
fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base
editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD
mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks,
Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced
sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal
haematological parameters and reduced splenic pathology compared to mice that received
unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene
editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-
HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human
HSPCs avoided the p53 activation and larger deletions that have been observed following
Cas9 nuclease treatment. These findings point towards a one-time autologous treatment
for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the
undesired consequences of double-strand DNA breaks.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021 Session-9: NON-VIRAL NUCLEIC ACID TRANSFER

AVINASH BAJAJ
Regional Center for Biotechnology, New Delhi, India

Nanoparticle-mediated gene therapy strategies for mitigating
inflammatory bowel disease

Nucleic acid (NA) therapeutics are emerging technologies to target the diseases of
gastrointestinal tract (GIT) like inflammatory bowel disease (IBD), but face major challenges
due to hydrophilic, negative charge, and degradable nature of nucleic acids in GIT. In my
talk, I will present our attempts to engineer lipid and polymer based nanogels for delivery
of therapeutic siRNA and plasmid DNA to the inflamed colon. Post-translational
modifications like SUMOylation have been shown to play a crucial role in IBD pathogenesis,
and polymeric nanoparticles are known to deliver the NA therapeutics to different cell
types. My talk will highlight the engineering of polymeric nanogels that can stabilize the
NAs in harsh gastrointestinal conditions. Oral delivery of these nanogels can target the
post-translational modifications in the GIT and mitigates the gut inflammation. I will also
present the engineering of lipid-based nanoparticles that can deliver the siRNA
therapeutics to the epithelial cells, macrophages, and dendritic cells. Oral delivery of these
siRNA entrapped lipid nanoparticles targeting of pro-inflammatory cytokines can mitigate
the gut inflammation, and inhibit the infiltration and polarization of CD4 T cells in inflamed
gut. In summary, I will present the recent progress of our group in the engineering of non-
viral mediated gene therapy vehicles for targeting IBD.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

CHANTAL PICHON
CNRS & University of Orléans, Orléans, France

Co-delivery of NS1 and BMP2 mRNAs for bone regeneration
Messenger RNA (mRNA) activated matrices (RAMs) are interesting for bone regeneration
since they allow in-situ and sustained production of osteogenic proteins. RNA sensors
activation and lacking of proper in vivo mRNA delivery vector are two main issues of
mRNA-based therapy. Inspired by viral immune evasion protein ability to inhibit host cell
responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A
virus as an mRNA enhancer. We evidenced a dose- dependent blocking of RNA sensors by
NS1 expression. Dual NS1 and BMP-2 mRNA delivery to murine pluripotent stem cells,
promoted osteogenic differentiation and extracellular mineralization. Next, we adapted our
histidylated lipopolyplex (LPR) platform for in vivo BMP2/NS1mRNA delivery in form of
RAM. SEM imaging demonstrated that loaded LPRs maintained their spherical morphology
in RAM, thanks to the core-shell structure of LPRs. mRNAs in vitro release from RAMs lasted
for 16 days and 2-fold more protein expression compared to cell transfection without
RAMs. Eight weeks following subcutaneous implantation in back of mice, microCT and
histology analyses showed that the BMP2/NS1 LPR containing RAMs induced significantly
more new bone tissue than those without NS1 mRNA. Overall, our results demonstrate that
BMP2/NS1 dual mRNAs system is suitable for osteogenic engagement, and freeze-dried
RAM-BMP2/NS1 mRNAs could be promising off-the-shelf product for clinical orthopedic
practice.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

PRIYA PRAKASH KARMALI
Arcturus Therapeutics, San Diego, USA
Self-transcribing and replicating RNA based vaccine for SARS-CoV-2
Arcturus is developing, a low dose self-replicating mRNA-based vaccine (ARCT-021) for
SARS-CoV-2. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-
length spike glycoprotein. The mRNA is delivered using LUNAR®, Arcturus’ proprietary
lipid nanoparticle-based delivery technology. Vaccination with ARCT-021 protected human
ACE2 transgenic mice from both mortality and measurable infection following wild-type
SARS-CoV-2 challenge. Furthermore, single administration of ARCT-021 was effective in
primate model with vaccinated macaques showing substantial reductions in median lung
viral titers. ARCT-021 has also demonstrated a favorable safety and immunogenicity profile
in the clinic. This presentation will provide a summary of development of the ARCT-021
vaccine including recent advancements.

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021

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

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

6th Annual Symposium on Cell & Gene Therapy, 1-3 September, 2021
Centre for Stem Cell Research (a unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, India