The document provides a comprehensive overview of gene therapy, detailing its types, methods, vectors, and major developments, as well as challenges faced in the field. It explains both somatic and germline gene therapies, the use of various viral and non-viral vectors, and notable case studies such as treatments for genetic disorders and cancers. Additionally, it addresses issues like immune response, safety concerns, and the history of significant developments, including the first approved gene therapy for severe combined immunodeficiency (SCID).

1. Gene Therapy- Types, Methods, Vectors, Major
Development, Problems
Sanju Kumari

2. • Gene therapy is the insertion of genes into an individual cells
and tissues to treat a disease in which a defective mutant
allele is replaced with a functional one
• DNA is used as a therapeutic agent
• Genetic diseases, hematological disorders, acquired
immunodeficiency syndromes, cancers are mainly treated

3. Types of gene therapy
Germ line gene therapy Somatic cell gene therapy

4. Somatic cell gene therapy
IN VIVO EX VIVO

5. Hematopoetic stem cell gene therapy

6. Embryonic stem cell gene therapy
• Neuronal degeneration
results into retinis
pigmentosa, retinal
detachment,glaucoma
• Neural stem cell may help to
restore vision in patient
• Epidermal growth factor or
fibroblast growth factor used
for proliferation and
maintenance invitro

7. Vectors for gene therapy
• Viral vectors • Non-Viral vectors
Retroviral vectors Oligonucleotides
Lentiviral vectors Naked-DNA
Adeno associated viral Polyplexes
vectors Lipoplexes
Adenoviral Vector Liposomes

8. RNA Virus
• Retrovirus -The enveloped virus particle contains two copies of
the viral RNA genome, surrounded by a cone-shaped core
• It is Integrating virus

9. Retroviral life cycle

10. • Separation of the cis and
trans functions of a
retrovirus in a recombinant,
replication defective vector
system
• Recombinant virus is made
by introducing all these
elements into the same cell
• SCID-X gene therapy

11. Production of Retroviral particle
(Verma & Weitzman, 2005)

12. • Lentivirus – It encodes three to six additional viral proteins,
which contribute to virus replication and persistence of infection
• Human immunodeficiency virus type 1 (HIV-1) - based vectors
most extensively studied
• Thalassemia treatment

13. DNA Virus
• Adenovirus – double stranded linear DNA
• Cause respiratory (especially common cold), intestinal and eye
infections in human
• It is non integrating virus
• Used for cancer therapy

14. Adenovirus

15. Adenoviral-mediated Rybp expression promotes tumor
cell-specific apoptosis
• Rybp kills tumors but not non transformed cell
• Ad-Rybp treatment result in Cytotoxic effect in the
osteosarcoma cell line U20S
• It enhances Fas-induced apoptosis
• Ad-Rybp infection sensitizes cells to TNF-α treatment

16. Ad-Rybp infection induces apoptosis

17. • Adeno - associated virus
 Single stranded DNA
 Episomal forms
 Deliver genes to brain, muscle, eye

18. • Treatment of hemophilia B
• Deficiency of factor IX
• Therapeutic delivery of the
canine factor IX gene to
hemophilic dogs.
• An efficient, non-
immunogenic
and persistent vector for
mediating therapeutic gene
delivery

19. Liposome
• Liposomes are
phospholipid vesicles (50 –
100 nm)
• They have a bilayer
membrane structure similar
to that of biological
membranes and an internal
aqueous phase
• Liposomes show excellent
circulation, penetration and
diffusion properties

20. Lipoplexes and polyplexes
• Plasmid DNA can be
covered with lipids in an
organized structure like a
micelle or a liposome
• When the organized
structure is complexed
with DNA it is called a
lipoplex
• Complexes of polymers
with DNA are called
polyplexes

21. Lipoplexes and polyplexes mediated transfection

22. Dendrimer
• These are highly branched
synthetic polymers (<15 nm)
• It show layered
architectures constituted of
a central core, an internal
region and numerous
terminal groups
• Wide application in Drug
Delivery System (DDS) and
gene delivery

23. Dendrimer delivery of an anti-VEGF oligonucleotide into
the eye
• Lipophilic amino - acid dendrimer is used to deliver an anti
-vascular endothelial growth factor (VEGF) oligonucleotide
(ODN-1) into the eyes of rats
(Marano et al., 2005)
• It inhibit laser - induced choroidal neovascularization (CNV)
Dendrimer used for ODN-1 delivery

24. Methods of gene transfer
• Physical methods :-
 Microinjection
 Electroporation
• Chemical methods:-
 Calcium phosphate precipitation

25. Femtosecond laser-assisted microinjection into living
neurons
• Femtosecond laser is used
to perforate vital cells
• Transfection efficiency
reached almost 100%.
• Advantage –
 contact-free
 non-disruptive
 stable transfection.

26. Optical damage of astrocyte irradiated by femtosecond laser

27. Electroporation
• Electroporation is the best non viral transfection technique in
human endothelial and smooth muscle cells
• High efficiency and acceptable survival rate
• Require special buffer and programs
• A range of voltage,capacitance and resistance settings is used
• High number of cells and high plasmid amounts required is a
weakness
(Iversen et.al.,2005)

28. Calcium phosphate transfection
• Most popular tools in neuroscience research
• Low cell toxicity and easiness to use
Fluorescent images of GFP-transfected cells

29. Chimeraplast
(Richardson et.al.,2002)

30. Triplex forming oligonuleotide
(Richardson et.al.,2002)

31. Small fragment homologus replacement
(Richardson et.al.,2002)

32. • Site-specific gene modification by oligodeoxynucleotides in
mouse bone marrow-derived mesenchymal stem cells
• Alternative approach to ‘cure’ genetic disorders caused by
mutations
• Establishement of MSCs cell lines with stably integrated mutant
neomycin resistance and enhanced green fluorescent protein
reporter genes
• The genetically modified MSCs were able to engraft into many
tissues of unconditioned transgenic mice
(Flagler,2008)

35. Sleeping beauty transposon system
• Transposon- used as
therapeutic agent for gene
transfer
• It has been used to
accomplish stable
chromosomal integration of
functionoing gene in somatic
cells of adult mice of the
factor IX for hemophilia
(Richardson et.al.,2002)

36. • Somatic integration and long-term transgene expression in
normal and hemophilic mice using a DNA transposon system
• Sleeping beauty transposase efficiently insert DNA into the
genomes of adult mammals using naked DNA
• Long-term expression of human blood coagulation factor IX
• Therapeutic in a mouse model of Haemophilia B
(Yant et.al.,2000)

37. Vectors for Sleeping
Genetic assay for
Beauty-mediated
transgene integration in
transposition
cultured cells.

38. Genetic assay used to recover transposons from
mouse chromosomes

39. Octaarginine-modified multifunctional envelope-type
nanoparticles for gene delivery
• Multifunctional envelope-type nanodevice (MEND) that mimics
an envelope-type virus based on a novel packaging strategy.
• DNA core packaged into a lipid envelope modified with an
octaarginine peptide
• Topical application of MEND particles containing constitutively
active bone morphogenetic protein (BMP)
• Type IA receptor (caBmpr1a) gene had a significant impact on
hair growth in vivo
• Superior non-viral gene delivery system
(Khalil et al.,2007)

40. The multifunctional Hair follicle formation in mice
envelope type nano skin treated with MEND3
device

41. Major Development
First Approved Gene Therapy
Ashanthi De Silva - A rare genetic
disease called severe combined
immunodeficiency (SCID)
Defective adenosine deaminase
gene results in deficiency of ADA
protein
It plays important role in
deamination reaction
Lack of healthy immune system Dr. W. French Anderson
with four-year old Ashanthi
De Silva at U.S. National
Institutes of Health

42. • Correction of SCID(X) by exvivo gene therapy
• Mutation in the gene encoding the common γc chain
• γc chain is an essential component of five cytokine receptors,
which are necessary for the development of T cells and natural
killer cells.
• CD34+ bone marrow cells from five boys are transduced ex
vivo
• Retroviral vector
(Abina et al.,2002)

43. Gene correction using retroviral vectors

44. No. of cells after gene Normal sized thymus
transfer

45. Repeat administration of DNA / liposomes to the nasal
epithelium of patients with cystic fibrosis
• CFTR is a cAMP-activated chloride channel in the apical
membrane of epithelial cells
• Loss of CFTR leads to impaired electrolyte movement in the
organs
• CFTR cDNA complexed with DC-Chol/DOPE cationic
liposomes
(Hyde et al.,2000)

46. CFTR immunohistochemical staining of nasal brushing cells

47. Third strand-mediated psoralen-induced correction
of the sickle cell mutation
• Plasmid containing a b-globin gene fragment transfected into
cells
Interaction of the psoralen delivery strand with the target
sequence

48. Screening assay for mutation correction
(Varganov et al., 2007)

49. Gene Therapy May Switch off Huntington Disease
• RNA interference or gene silencing may be a new way to treat
Huntington's
• siRNA is designed to match the RNA copied from a faulty gene
• HD results from polyglutamine repeat expansion (CAG codon)
in exon of huntingtin gene
• Toxic gain of function on the huntingtin protein
(Harper et al.,2005)

50. RNAi expression to
Human htt expression by mouse striatum
RNAi

51. Gene Therapy Tackles Blood Disorder
• Blood disorder Thalassemia
• Additional splice site is formed due to mutation
• Repairs errors in messenger RNA derived from defective
genes
Correction of aberrant splicing by modified U7 snRNAs

52. Correction of aberrant splicing by U7.623 snRNA in -globin IVS2
mutant cells
(Vacek et al., 2003)

53. Dystrophin expression in mice by intravascular injection of
naked DNA
Duchenne muscular dystrophy (DMD) - lethal, X-
linked,recessive disease caused by a defect in the dystrophin
gene
Injection of DNA solution by tail Effect of histamine on gene
artery and tail vein expression

54. Gene Therapy for Parkinson's disease
• Liposomes coated in a polymer call polyethylene glycol (PEG)
• Viral vectors are too big to get across the "blood-brain barrier"
This method has potential for treating Parkinson's disease Loss
of dopaminergic neurons
• Tyrosine hydroxylase gene therapy
• Episomal based gene therapy
(Pardridge et al., 2005)

55. A super-coiled expression plasmid DNA encapsulated in an 85 nm
pegylated PIL targeted to a cell membrane receptor (R) with a
receptor-specific, endocytosing mAb
(Pardridge et al.,2005)

57. First Deafness Cure in guinea pig
• Destruction of the hair cells in the cochlea that translate sound
vibrations into nerve signals
• A gene, called Atoh1, which stimulates the hair cells' growth,
was delivered to the cochlea by an adenovirus
• Regained up to 80% of their original hearing thresholds
(Kawamoto et al.,2005)

58. Gene Therapy for Advanced Melanoma
• Reengineer lymphocytes for expression of TCR to target and
attack cancer cells in patients with advanced metastatic
melanoma
• Retroviral vector was constructed encoding the pmel-1 TCR
genes targeting the B16 melanoma antigen, gp100
• Adoptive cell transfer
• Transduction of C57BL/6 lymphocytes resulted in efficient
pmel-1 TCR expression

59. • Objective -
The most severe forms of inherited blindness are collectively known
as Leber congenital amaurosis (LCA) and are the first type of
inherited blindness to be treated by gene therapy

60. Materials and Methods
• A recombinant AAV-2 vector was used to deliver a human
RPE65 cDNA to the target retinal pigment epithelium cells
• Maguire et al.used an AAV-2 vector with a constitutive
promoter to drive transgene expression
• Bainbridge et al.used elements of the endogenous RPE65
promoter
• detected a progressive improvement in retinal sensitivity
in one patient using both microperimetry and dark-adapted
perimetry

62. Results

63. Assesment of visual mobility

64. Problems in gene therapy
• Short-lived nature of gene therapy
• Immune response and toxicity
• Problems with viral vectors
• Restricted targeting of specific cell types
• Multigene disorders
• Insertional mutagenesis
• Religious concern
• Deaths may occured

65. Failures of Viral Mediated Gene Therapy
• Retroviral vector
• Dr. Alan Fischer – Conducting gene therapy on SCID-X1
linked hereditary disorder
• Hematopoietic stem cells from patients were stimulated and
transduced ex vivo with MLV-based retroviral vector
• Expressing the γc cytokine receptor subunit, and then were
reinfused into the patients
• During a 10-month follow up, γ c-expressing T and NK cells
counts and function were comparable to age-matched
controls
• Two of the children developed T-cell leukemia

66.  Adeno-Associated Virus Vector
• Patients suffering from hemophilia B were treated with AAV
vectors expressing human factor IX
• Intramuscular injecting AAV factor IX vectors directly into
liver, which in turn have shown some unexplained toxicity
 University of Pennsylvania
• A human Phase I clinical trial for ornithine
transcarbamylase deficiencies
• This trial was designed to test the safety of an E1/E4-
deleted recombinant adenovirus vector
• Jessie Gelsinger received highest dose and first person to
die as result of vector delivery