The document summarizes gene therapy approaches for two monogenic diseases: ADA-SCID and cystic fibrosis. For ADA-SCID, early clinical trials involved ex vivo gene transfer to T-cells or hematopoietic stem cells with retroviruses, showing long-term expression in some patients. For cystic fibrosis, various delivery vectors like adenovirus and liposomes were tested in preclinical and clinical trials via nasal or lung administration, with some trials demonstrating transient CFTR expression and correction in a fraction of patients. Overall, the effectiveness of gene therapy for these diseases depends on developing safer and more efficient delivery methods to achieve sustained therapeutic levels of expression.

1. GENE THERAPY FOR MONOGENIC DISEASES

2. ADA DEFICIENCY
• Irreversible deamination of adenosine to inosine and 2-
deoxyadenosine
• Detoxification reaction
• Deficiency results in SCID
• Accumulation of RBX dATP, plasma and urinary dAdo,
decreased levels of SAHH
• Toxic to B/T cells
• 1st immunodeficiency with identified underlying molecular defect
• BMT
• Extracellular enzyme replacement therapy (PEG-ADA)

3. GT FOR ADA-SCID
• Monogenic disease
• Well defined target tissue and cells
• Easy manipulation of HSCs
• Ex-vivo delivery of ADA gene
• Genetically modified cells have advantage over defective cells
• Small size of cDNA (1.1Kb) to be cloned
• Easy cloning in a variety of vectors
• ADA gene does not need cell-selective promoter
• Even a fraction of transfected cell population with as little as
10% of normal expression can complement immune function

4. PRE-CLINICAL STUDIES
Gene transfer to HSCs
• Integration of transgene
• Transfer to all daughter cells
• Once-only procedure
• Difficult to isolate, culture and transduce
1. Studies in murine models
• Successful transfer and long term expression
2. Redo same experiments in larger animals
• Short term expression of human transgene
3. Transduction of human HSCs
• Bone marrow culture transfected by retrovirus
Use of CD34+ cells, umbilical cord HSCs

5. Gene transfer to T cells
• Use of patient derived T-cells. In-vitro retroviral transduction
• Long term survival
• In vivo injection of T-cells from ADA-SCID patients to
immunodeficient mice
• Reconstitution of immune function
PRE-CLINICAL STUDIES

6. CLINICAL TRIALS

7. T-CELL GT
• Two girls in 1990 (already on PEG-ADA)
• Infection of patient T-cells with retrovirus containing ADA cDNA
+ rIL-2 and an anti-CD3 antibody
• Ex-vivo transfer to patient after 9-12 days
• 12 times injection for 18-24 months
• Transduction efficiency 0.1-10%
• Stable expression after 2 years

8. T-CELL DEPLETED BM AND PERIPHERAL BLOOD
LYMPHOCYTE GT
• Similar patients
• Infusion of transduced T-cells
• Followed by infusion of transduced HSCs
• Different vectors
• Over 2 years
• For 1 year, vector positive cells were from T-cells
• Afterwards from BM origin
• Better but low immune response

9. CD34+ CELLS GT
• Retroviral mediated gene transfer to BM CD34+ cells in 3
children
• 5-12% cells transfected in vitro
• Tranduced cells present in patients but no expression
• Umbilical cord CD34+ cells in three infants

10. GT FOR CYSTIC FIBROSIS

11. • AR disease (1/2500 live births)
• Diagnosed at birth or early childhood
• Respiratory, intestinal, fertility symptoms
• Use of antibiotics/physiotherapy-increased life expectancy
• CFTR gene
• Reduced mucosal clearance+reduced defense against
pathogens

12. GT
• Difference b/w DAD-SCID and CF
• As low as 5% expression is sufficient
• Development of delivery systems and protocols

13. DELIVERY VECTORS FOR CF
Adenovirus
• Natural tropism for lungs
• Infect non-dividing cells
• Can be produced at high titres
• Wild type AV have minor pathologies
• Promising in-vitro results
• In-vivo studies in mice (KO)
• Human CFTR gene expression in cotton rats and primates

14. Cationic Liposomes
• liposome-DNA complex
• Lung administration as aerosols or direct lavage
• Intravenous injection?
• Non-toxic, nonimmunogenic, no delivery to gonads
• Relatively inefficient
DELIVERY VECTORS FOR CF

15. CF CLINICAL TRIALS
Nasal administration
• Ideal site
• 1st study in 1993 in three CF patients (AV mediated)
• Local inflammation
• Expression in 2 patients for 10 days
• 2nd trial (AV mediated)
• 30% expression for 2 weeks
• Blind-placebo controlled trial in 12 patients (AV mediated)
• Expression in 5/6 patients but inconsistent
• A double blind-placebo controlled liposome mediated trial
• Expression in 5/8 patients, 20% channel correction for 7
days

16. Administration to LRT
• Delivery of CFTR cDNA to lungs by bronchoscope
• One sample had normal CFTR protein
• Inflammatory reaction
• Aerosol administration by a rAV
• Expression of DNA but no data on ion transport improvement
CF CLINICAL TRIALS

17. CONCLUSION
• ~50% samples have transgene expression and 30% show functional
correction
• Safe for nasal delivery and dependent on dose for lungs
• Crucial importance of delivery method
• Nebulization