related to gene therapy. Resources used are uptodate by wolter kluvers and Harrison Principle of Internal Medicine,20e

1. GENE BASED AND CELL
BASED THERAPY IN
CLINICAL MEDICINE
Moderator : Dr.T.Murali Venkateswara Rao
Presenter : Dr.Puppala Santosh

2. INTRODUCTION
• Genetic diseases including, Inherited diseases
(Mendelian/Mitochondrial/Polygenic) or acquired
diseases(E.g. Cancers, Infections), a number of
methods of treatment are under development.
• Like Enzyme replacements, Monoclonal antibodies,…
• But the recent approach was changing or silencing
the genetic sequences that led to disease.

3. GENE THERAPY
• Novel area of therapeutics where GENE TRANSFER is used
to treat the disease.
• Refers to introduction of EXOGENOUS GENE(S) into one or
more autologous or allogenic cell types.
• Gene based or Cell based therapy are overlapping fields of
Biomedical research with same therapeutic goal.
❑Manipulating Patient cells using genetic material-
GENE(BASED) THERAPY
❑Infusing or transplanting whole cells- CELL BASED
THERAPY

5. Technique of GENE TRANSFER

6. GENE TRANSFER
• It is transfer of nucleic acid sequence.
• It has 3 Essential Elements:
1. Vector or Gene delivery Vehicle
➢Delivery of Naked DNA or RNA to a cell is inefficient.
➢Usually Engineered from Virus by deleting some or all viral
genome
2. Transgene
➢Therapeutic gene of interest.
3. Target Cell
➢Cell to which transgene is delivered.

7. TRANSDUCTION
• The series of steps in which the vector and donated DNA
enter the target cell and express the transgene.
• Cells can be transduced either in vivo (or) in vitro.
❑In vivo
➢Literally means inside of the body.
➢Vector is directly is injected in to the patient.
➢HSC can be transfused in vivo.
➢In vitro
➢Literally means outside of the body.
➢Removal of target cell from the patient, manipulating the gene in the
lab followed by return of the gene-modified autologous cells to the
patient
➢ better technique.

8. In vivoIn vitro

9. VECTORS OR GENE DELIVERY
VEHICLES
1.Definition
2.Characteristics of Vectors
3.Strategies to reduce immunogenicity.

10. VECTORS-DEFINITION
• Constructs(genes modified) for gene therapy are delivered to
target cell using the vectors.
• Choice of Vector has major impact on the efficacy and safety
of the therapy.
• Most of the vectors are derived from derived from viruses,
although other type of vectors can be used (plasmids,
artificial chromosomes, nanoparticles).

11. VECTORS-CHARACTERISTICS
1. CAPACITY – Different Vectors accommodate different sized constructs.
2. PRODUCTION – need to be produced in sufficient quantities. Production
demand may influence the vector selection.
3. TARGET CELLS – some viruses has tissue tropism like some viruses infect
dividing cells and some quiescent cells.
4. EXPRESSION LEVEL – some genes require higher expression level, but some
sufficient at low levels. For say, In AR inheritance restoration of small amount of
gene function is sufficient.
5. DURATION OF EXPRESSION – Long term expression(in gene deficiencies)
vs transient expression(Cancers)
6. IMMUNOGENICITY – virus infection is common in general population and it
may limit the use of certain viruses as the host immune response leads to side
effects and loss of potency
7. INTEGRATING VS NON INTEGRATING

12. VECTORS – INTEGRATING Vs
NON INTEGRATING
• INTEGRATING VECTORS
➢Insert the construct into the genomic DNA of host cells and
replicate at every cell division.
➢Associated with risks of integration like New fusion genes,
damaging existing genes, leading to increased expression of
adjacent genes
➢Initial reports with integrating vectors described cases of
oncogenesis, including fatal leukemogenesis.
➢Retroviruses and Lentiviruses.

13. VECTORS – INTEGRATING Vs
NON INTEGRATING
• NON INTEGRATING VECTORS
➢Vector DNA remain episomal (non integrated with host DNA) and will
be eliminated when the cell divides or dies.
➢Derived from non integration viruses like Adenovirus(AV), adeno-
associated virus(AAV),Vaccinia virus, integration-deficient lentivirus,
non-viral vectors (plasmids, artificial chromosomes, nanoparticles).
➢ Avoid oncogenic side effects but concerns about very low frequency
integration of AAV into cellular DNA and potential risk of genotoxicity.
➢Another disadvantage with AV is tropism to liver and their
immunogenicity. The potential consequences are:
✓Inability to receive gene therapy due to prior immunity
✓Immune/inflammatory Complications of treatment.

15. VECTORS-STRATEGIES TO REDUCE
IMMUNOGENICITY(for Adenoviral Vectors)
1. Use of Adenovirus serotypes that most people not exposed
▪ Group B Adenovirus serotype 35
2. Use of Adenovirus serotypes that infect other species
3. Insertion of genes that inhibit host immune response
▪ Genes of Early region 3[E3] from adenovirus
4. Deletion of Viral genes that create immunogenic epitopes
5. Induction of Immune tolerance

16. RECOMBINANT
AAV VECTORS
• Emerged as attractive gene delivery
vehicles because of its long term
expression in genetic diseases.
• Engineered from a small replication-
defective DNA virus, devoid of viral
coding sequences.
• Trigger very little immune
responses in experimental animals.
• Capable of transducing non dividing
target cells, donated DNA is
stabilised in episomal form, thus no
complications related to insertional
mutagenesis.
• It has tropism to certain long lived
cells(Skeletal muscle, CNS,
hepatocytes) and long term
expression can be achieved even in
absence of cell division.

17. FIRST LICENSED PRODUCT
❑ In Europe
✓For AR LPL deficiency
✓Intra-muscular injection of AAV-LPL .
❑In USA
✓For retinal degenerative disease LCA
✓Subretinal injection ofvAAV-RPE65

18. CLINICAL APPLICATIONS
OF GENE THERAPY
❑ Gene therapy is a different approach
when compared with other
therapeutics, in that it aims to treat
the underlying genetic cause of the
disease.
❑ Most common indication of gene
therapy is Cancer followed by
genetic disorders.

19. GENE THERAPY FOR
GENETIC DISEASES
Gene therapy is an appealing way to provide a normally
functioning gene to an individual who has inherited a
pathogenic gene variant(s).
I. Immunodeficiency disorders-SCID
II. Neurodegenerative diseases-ALD,MCLD
III.Haemophilia
IV.Retinal gene therapy

20. David Vetter
(Bubble Boy)

21. I. IMMUNODEFICIENCY DISORDERS
• First therapeutic effect from gene transfer occurred with X-linked SCID,
which is due to mutation in gene IL2RG encoding the Ύc subunit of
cytokine receptors.
• Transduced cells have proliferative advantage even in small number
compared with Non transduced cells which don't have maturation of
lymphocytes.
• Complete reconstitution of the immune system and remarkable gains in
the growth occurred, up to 17 years of complication free life(in 15
members).
• 18 of the 20 children has achieved in correction in immunodeficient status.

22. 1. IMMUNODEFICIENCY DISORDERS
• But 5 of the 20 developed T cell ALL and is due expression of
LMO-2 gene after the integration of the transgene by the
retroviral vector (Insertional mutagenesis)
• Use of Suicide gene cassette or Insulator gene cassette in
vectors, use of lentiviral vectors rather than retroviral
vectors are some of the strategies used to overcome the
complication.

23. 1. IMMUNODEFICIENCY DISORDERS
• More clear-cut success has been achieved in the another form of
SCID,ADA-SCID.
❑It can be treated with enzyme replacement therapy using PEG-ADA which costs
around $200k-$300k per annum.
❑It leads to immune reconstitution but not always to normal T cell count.
❑Initial trails were unsuccessful but later with some modifications became
successful.
❑Using Milan protocol, there have been no complications for 10 children with
median follow-up of age >11 years.
❑In 2016, it was approved by European Medicines Agency.

24. 2. NEURODEGENERATIVE DISEASES
• SCID trails supported the hypothesis that gene transfer can
be done in any disease for which ABMT is therapeutic.
• Cartier and Aubourg conducted first trail of X-linked
adrenoleukodystrophy, a neurodegenerative disorder.
• Used Autologous Lentiviral vector transduced HSCs
• Dramatic stabilisation occurred in the disease.
• It showed that stem cell transduction also works for
neurodegenerative diseases.
Contd…..

25. 2. NEURODEGENERATIVE DISEASES
• Investigators in Milan developed a treatment for
Metachromatic Leukodystrophy, neurodegenerative disorder,
type of LSD.
• A Lentiviral vector that directed supraphysiologic levels of
ARSA expression in the transduced cells was developed.
• Transduction of these autologous HSCs to children
presymptomatically, led to preservation and continued
acquisition of motor and cognitive milestones as long as 32
months after the effected sibling has lost the mile stones.
• This illustrates the level of expression can allow gene
therapy to succeed.

26. TRANSDUCTION OF HSCs TO TREAT
OTHER DISEASES
❑Hemoglobinopathies
▪ Studies already conducted
▪ Higher hurdle in terms of extent of transduction required to achieve
therapeutic effect.
❑Many haematological disorders are under trail
▪ Sickle cell disease
▪ Thalassemia
▪ Wiskott-Aldrich Syndrome
▪ Chronic granulomatous disease

27. 3. HAEMOPHILIA
• Considered a promising disease model for gene transfer
because
o Gene product doesn't require precise regulation
o Biologically active clotting factors can be synthesised in a variety of
tissues.
• Circulating factor levels from <1% into a range of 5% greatly
improves phenotype of disease.
• In Haemophilic dog models in preclinical studies,
recombinant AAV vectors infused into skeletal muscle or liver
resulted in Long-term (>5 years) expression of Factors VIII &
IX.
Contd…..

28. 3. HAEMOPHILIA
• First trails of an AAV vectors expressing F-IX delivered to liver in
humans with Haemophilia B.
➢It resulted in therapeutic circulating levels but for only 6-10 weeks.
➢Due to memory T cell response to nucleocapsid of virus.
• In Second trail, a short course of prednisolone was administered
when F-IX decreased.
➢It resulted in long term (>7 years) expression of F-IX.
➢It is seen in 2-7% with severe haemophilia in men.
• By using high specific activity of variant of F-IX transgene
reduces vector dose required and decrease in immune response
finally leading to increased plateau levels of F-IX.
• Current efforts are being directed towards Haemophilia A.

29. 4. RETINAL GENE THERAPY
• Haemophilic trails proved that avoidance of immune
response is key for Long term expression.
• Thus immunoprivileged sites such as retina began the
therapeutic targets.
• This inference was confirmed in treating retinal
degenerative disease Leber congenital amaurosis(LCA).
➢Animal models which are treated subretinal injection of AAV
vectors expressing RPE65 transgene before 10 years shows electro
retinal and behavioural evidence of visual function.
➢Phase III trails in humans are completed and results showed
improvement in multiple measures of retinal and visual functions.

30. 4. RETINAL GENE THERAPY
• It was approved by FDA and is the first licensed product in
USA.
• Trails are being done for Choroideremia,Retinitis
pigmentosa (Inherited retinal degenerative disorders) ,Age-
related macular degeneration (a acquired disorder).

31. GENE THERAPY FOR
CANCER
Majority of clinical gene transfer subjects are with cancer. The
intention was to increase the precision in therapies and make
them less Toxic and more effective. The approaches are:
I. Modifying the cancer
II. Modifying the host
III.Combination approaches-Modification of Host and
Tumour by Virotherapy
IV.Other approaches

32. 1. MODIFYING THE CANCER
Being a acquired genetic disorder, initial trails are directed at
correcting genetic defects or introducing lethal genes.
1. Tumour Correction
✓ Intratumoral approach with Adenoviral-mediated expression of tumour
suppressor gene p53.
✓ Initial studies showed some complete and partial responses in SCC of Head and
neck, oesophageal cancer, NSCLC.
✓ Yet no licensed product except in china.
2. Pro-Drug metabolizing genes
✓ Introduction of prodrug or suicide gene that increases sensitivity to cytotoxic
drugs.
✓ Adenoviral vector expressing Thymidine kinase (TK) gene transduction.
✓ Cells that expresses TK can be killed with ganciclovir.
✓ This is examined in aggressive brain tumours and locally recurrent prostate,
breast and colon tumours.

33. 2. MODIFYING THE HOST
1. Immunological Modifications
i. Recruiting the immune system.
➢Monoclonal antibodies have been successful in activating anti tumour activity
of the immune response.
➢Use of construct that produces recombinant monoclonal antobody is under
investigation and trails
ii. Vaccination
➢Sipuleucel-T, a dendritic cell vaccine for treatment of recurrent prostate
cancer has been approved in USA
iii. Adoptive immunity
➢Chimeric antigen Receptor T- Cells targeted against CD19 in intractable B-
ALL have produced >90% complete response rate.

34. CHIMERIC ANTIGEN RECEPTORS
• Isolating T cells from patients
and re-engineer them to
express CARs that recognise
antigens present on the
Tumour cells.
• Significant issues are Cytokine
release syndrome, Organ
Toxicity , Neurotoxicity and
needed aggressive support and
care to patients.

35. 2. MODIFYING THE HOST
II. Non Immunological Modifications
➢Gene transfer can be used to protect normal cells from toxicities of
chemotherapies.
➢Most extensively studied approach is to transduce hemopoietic cells
with genes encoding resistance to Chemotherapeutic agents,
including MDRI or MGMT.
➢Studies which are in early phase are gene transfer to inhibit host
angiogenesis by constitutive expression of inhibitors like angiostatin
and endostatin ,or transfer of T cells that are genetically modified to
recognise specific antigens of newly formed vasculature.

36. 3. COMBINATION APPROACH-
VIROTHERAPY
IMMUNO ONCOLYTIC VIRUSES
• These viruses are genetically modified to replicate in the tumour
cells but not in normal cells.
• Talimogene Laherparepvec (T-Vec) has got approval recently by
FDA for treatment of melanoma.
• It is a oncolytic herpes virus containing human GM-CSF gene.
• Physical limitation to virus spread including fibrosis, basement
membranes, necrotic areas in tumour decreases the efficacy of virus.
• Activity against the metastatic disease was limited.

37. 4. OTHER APPROACHES
• Till now we focussed on gene addition therpy.
• Another new technique is under development, that is
GENOME EDITING.
✓Mutation is corrected in-situ
✓It uses novel reagents like Zinc finger nucleases,TALENs and
CRISPR.
• Another new strategy is introduction of siRNAs or short
hairpin RNAs as transgenes to knockdown the expression of
deleterious gene
✓E.g. Mutant huntingtin in huntingtons disease, genes of Hepatitis C
genome in infected individuals.

38. SUMMARY
• Gene therapeutics entered the
clinical testing in 1990s.
• Having a good clinical success,
it becomes one of the most
important therapeutic
modality of the 21st centaury.
• The concern is on long term
safety of this therapy and
regulating agencies mandate a
15-year follow-up for subjects
enrolled for gene therapy.

39. THANK YOU
Emmanuelle Charpentier Jennifer Doudna
Awarded Nobel prize in 2020 in chemistry for
CRISPR, the gene-editing tool