Gene therapy, strategies, vectors, applications

1. Ex vivo methods
Akash Mahadev Iyer
S-4 M.Sc Biochemistry
Department of Biochemistry
University of Kerala

2. • Gene ; entire nucleic acid sequence that is necessary for the synthesis of a
functional gene product (polypeptide or RNA).
 When there is a mutation in the gene, then it will change the codon, which will
change which amino acid is called for which will change the conformation of the
protein which will change the function of the protein.
 Genetic disorders result from mutations in the genome.
• Protein function determines, in turn, the cell phenotype and cell function. When
genes are changed in such a way that the proteins encoded by them are unable
to perform their normal function, a genetic disorder may occur.

3. • Gene therapy is a technique for correcting defective genes responsible for disease
development.
• Treatment of diseases caused by single gene recessive disorders- Monogenic
disorders (like cystic fibrosis, hemophilia, muscular dystrophy, sickle cell anemia
etc).

4.  Ashanti de Silva (4 years old) with severe combined immunodeficiency (SCID)
treated in 1990 at NIH in Maryland
 Lacked functioning immune system because of a defect in gene called adenosine
deaminase (ADA), which is involved in metabolism of dATP (nucleotide precursor
used for DNA synthesis)
 Accumulations of dATP are toxic to T cells
 Normal gene cloned into vector introduced into nonpathogenic retrovirus.

6.  An abnormal gene could be swapped for a normal gene through homologous
recombination.
 The abnormal gene could be repaired through selective reverse mutation, which
returns the gene to its normal function.
 The regulation (the degree to which a gene is turned on and off) of a particular
gene could b altered- Anti sense therapy, RNAi and Ribozymes.
Targeted Gene Mutation Correction
Targeted Killing of Specific Cells
Gene Augmentation Therapy (GAT)
Targeted Inhibition of Gene Expression

8.  Identify the gene(s) responsible for the disorder.
 Make copies of the normal gene.
 Insert the copies into vectors.
 “Infect” the affected cells with the vectors.
 Activate the gene so that transcription and translation take place
• Therapeutic molecule (small regulatory RNA or RNA- Antisense
oligonucleotides, aptamer, ribozymes, siRNAs), rapidly eliminated from body
• This can be counteracted by- chemical modification of phosphate backbone or
ribose sugars of these molecules, conjugation with specific ligands (PEG/
Cholesterol), association with a cationic lipid or polymer carriers.
Persistence of the therapeutic gene

9. GENE THERAPY
Germ Line Gene Therapy Somatic Cell Gene Therapy
Vector type
Non viral Viral
Lipid based Peptide based Polymer based
Naked Plasmid
Electroporation
Ballistic DNA
Sonoporation
Photoporation
Hydroporation
(Hydrodynamics)
Magnetofection
ESCsSperm cells Egg cells
Inorganic
Particles
Chemical Physical
Nuclei Pronuclear
Micro injection
Nucleic acid based
DNA/Cationic lipid
(Lipoplexes),
DNA/cationic polymer
(Polyplexes) and
DNA/cationic Polymer/cationic
Lipid (Lipopolyplexes)
Synthetic
Natural
Polyethylene imine (PEI), Dendrimers,
and Polyphosphoesters.
Proteins, Peptides,
Polysaccharides.
CaSO4, Au, QDs, CNTs, fullerenes,
supra molecular systems

10. SOMATIC CELL GENE THERAPY GERM LINE GENE THERAPY
Therapeutic genes transferred into the
somatic cells.
Therapeutic genes transferred into the germ
cells.
E.g.. Introduction of genes into bone
marrow cells, blood cells, skin cells etc.
E.g.. Genes introduced into eggs and
sperms.
Will not be inherited later generations. It is heritable and passed on to later
generations.
At present all researches directed to correct
genetic defects in somatic cells.
For safety, ethical and technical reasons, it
is not being attempted at present

11. Cells removed from body
Transgene delivered
Cells cultured
Cells returned to the body
Ex Vivo In Vivo
Transgene delivered
directly into host
Strategies for Transgene Delivery

12.  In ex vivo therapy (ex; out of, vivo; something alive), cells from person with a diseased
condition are removed from the patient, transformed and cells are reintroduced into the
patient
 Transfer of cloned genes into cells grown in culture.
 Transformed cells are selected, expanded by cell culture in vitro, then introduced into the
patient.
 To avoid immune system rejection of the introduced cells, autologous cells are normally
used (the cells are collected initially from the patient to be treated and grown in culture
before being re-introduced into the same individual.)
 Only applicable to tissues that can be removed from the body, altered genetically and
returned to the patient where they will engraft and survive for a long period of time (e.g. cells
of the hematopoietic system, skin cells, etc.)
 Therapeutic genes are delivered using vectors

13. • Ex- Vivo gene therapy involves transfer of genes in cultured cells which are then
reintroduced into the patient
• Steps
1. Isolation of cells (selected tissues e.g., Bone marrow) with genetic defect from a
patient
2. Growing the cells in a culture
3. Introduction of the therapeutic gene to correct the defective gene
4. Selection of the genetically corrected cells.
5. Transplantation of the modified cells to patient
 As this therapy involves the use of patient own cells for culture and genetic
correction, no adverse immunological reactions

15. • To transfer the desired gene into a target cell, a carrier is required. Such vehicles
of gene delivery are known as vectors
 Viral Vectors
 Non viral Vectors; engineered vectors- Naked DNA, particle based and
chemical based
Nonviral gene delivery methods
 Chemical and
 Physical approaches.
• The chemical methods include certain polymers and lipids, while the physical
methods utilize physical properties and forces to transport genetic material into
cells

17.  Integrate the gene in the cells.
 Activate the gene.
 It should protect the transgene against degradation by nucleases in the
extracellular matrix
 Easy and reproducible
 Target the right cells
 Production in high conc. per mL
 Avoid harmful side effects.
 Long term gene expression through insertion into the genome or stable episomal
persistence
 Controllable gene expression
 Tissue specific expression
 Low immunogenecity.

18.  Most common type of vectors -genetically altered to carry normal human DNA.
 Viruses replicate by inserting their DNA into a host cell.
 Viruses insert their DNA into cells with high efficiency.
 The most commonly used viral vectors are derived from retrovirus, adenovirus,
and adeno associated virus (AAV).
 Other viral vectors that have been less extensively used are derived from herpes
simplex virus 1 (HSV-1), vaccinia virus, or baculovirus.
The main drawbacks of using virus vectors are its immunogenicity and
cytotoxicity.
Viral vectors
DNA Viruses
Adenovirus, HSV, AAV,
Poxvirus, Alphavirus
RNA Viruses
Retrovirus,
(Lentivirus)

19.  dsDNA viruses -infect humans and other vertebrates.
 Cause benign infections of URT in humans
 Non-enveloped, does not integrate into the host genome
 Replicates as an episomal element in the nucleus
 Gene expression is short term and transient-repeated administration for
sustained expression
 Adenoviral vectors have been used to transfer genes in vivo into the lung, liver,
muscle, blood vessel, synovium, eye, peritoneum, brain, and tumors in animals
 Capsid proteins –Inflammatory response
 Nononcogenic (i.e., they do not cause tumors).
 Relatively easy to culture and
 Produced in large quantities.

21.  small non pathogenic, non env.ssDNA (4.7 kb long) virus
 A defective or “satellite” virus -depends on adenovirus (or some herpes viruses)
 Naturally replication-deficient virus,
 Replication is achieved via a helper virus (adenovirus or herpesvirus)
 Found in cells that are infected with adenovirus.
 Non-inflammatory to the host.
 Small size -allows it to penetrate many body tissues.
 AAV integrates its DNA into a single site in the genome of animal cells (the AAVS1
site on chromosome 19 in humans). -therapeutic gene to be permanently
integrated.
 Advantage- Replication-defective, nonpathogenic, and non immunogenic.
 Limitation- small transgene size (4681 nucleotides of single-stranded DNA)
 The virus can carry only a relatively short segment of DNA and they require helper
viruses for activation, Possible insertional mutagenesis .

23.  2 copies of a positive single-stranded RNA genome of 7 to 10kb.
 Enveloped virus ,deliver up to 8000 bases of ss RNA -yield permanent transduction
(delivered genes will be present inside the cell for the remainder of its life).
 When delivering RNA into cells via retroviruses, RT of the RNA to cDNA takes place via the
enzyme RT.
 cDNA integrates into the host genome via the enzyme integrase.
 Once integrated into the genome, it will be replicated when the cell replicates, -both of the
daughter cells will have a copy of the virally delivered gene. -“permanent transduction”.

24. • Random Integration via integrase -cDNA can be inserted in the middle of a
housekeeping gene. -renders the housekeeping gene useless and would lower
the amount of an essential protein, -leading to cell death
• If the cDNA were inserted into the middle of a tumor suppressor gene, -leads to
transformation of the cell into a tumor cell.
• If the cDNA were inserted upstream of a proto-oncogene, it could serve to
activate or mutate the gene into an oncogene, which will transform the cell.

25. Advantages
Non-viral methods
Injection of Naked DNA
Physical Methods to Enhance
Delivery
Electroporation
Gene Gun
Sonoporation
Magnetofection
Hydrodynamic delivery
Chemical Methods to enhance
Delivery
Oligonucleotides
Lipoplexes
Polymersomes
Polyplexes
Dendrimers
Inorganic Nanoparticles
Cell-penetrating peptides
Non-toxic
No immune response

26.  It involves chemical and physical methods such as direct injection of naked plasmid DNA
(particle bombardment), receptor-mediated endocytosis and gene transfer through
liposomes, polymers, nanoparticles etc.
 Not efficient as viral vectors,
Nonviral systems are attractive for the following reasons
 Very flexible with respect to size of DNA that has to be transported;
 They are cost-effective and easily allow large-scale production of plasmid DNA;
 They implicate less laborious safety testing as compared to recombinant vectors;
 There is no restriction on DNA insert length (although transfection efficiency decreases with
increasing DNA length).
Disadvantages
 Low gene transfer efficiency in vivo
 Transient expression due to lack of integration into the host genome;
 Immunostimulatory properties of plasmid DNA;
 In vivo toxicity due to accumulation of lipid components.

27. • Liposomes -hollow microscopic spheres of phospholipids, and can be filled with DNA or
other molecules during assembly.
• The liposomes will merge with the membranes surrounding most animal cells and the
contents of liposome end up inside the cell - lipofection .
• Nonspecific –they tend to merge with the membranes of any cell.
• Because of the aqueous environment inside cells and tissues, the hydrophobic tails of the
lipids will coalesce to form hollow liposomes, the interiors of which can contain
oligonucleotides for cellular delivery.

28.  Cationic polymers form stable polyplexes (nanosize polymer-DNA complexes)
with the desired DNA.
 Cationic polymer mediated gene delivery involves DNA complexation, complex
mediated transversion of cell membrane to the cytoplasm, release of DNA,
transfer of DNA into the nucleus
 PEI (Polyethylenimine), DEAE-dextran (Dimethylaminoethyl-dextran), PLL (Poly-
L-lysine) , Chitosan ,PPE (Polyphosphoesters)
 Inorganic nanoparticles of metallic elements, inorganic salts, or ceramics
produce complexes of size 10-100 nm.

30. The positively charged head group binds with negatively charged phosphate
group in nucleic acids and form uniquely compacted structure called lipoplexes.
Eg; DOGS (Dioctadecylamidoglycylspermine), DC-Chol (Cholesteryl 3β-N-(di-
methylaminoethyl)carbamate hydrochloride), bis-guanidium-tren-cholesterol
(BGTC)

32. Dendrimers is an artificially manufactured or synthesized molecule built up from
branched units called monomers.
Tree -like structure with defined molecular weights and entrapment properties
Comprises a core, layers of branched repeat units emerging from the core, and
functional end groups on the outside layer of repeat units

34. Gold NPs

36. Physical Methods to Enhance Delivery

37. Magnetofection

38. • The basic concept of using prodrug-converting enzymes is to limit the action of a
known cytotoxic drug to local tumor areas.
• Targeted prodrug therapy includes the delivery of a gene that activates a nontoxic
prodrug to a cytotoxic product by using viral vectors.
• This method maximizes toxicity at site of vector delivery while minimizing toxic
effects on distant cells.
• The cDNA of the enzyme is delivered in to the tumor by a vector.
• The corresponding nontoxic prodrug is applied and is taken up by tumor cells.
• Since these cells have incorporated the cDNA in to their genome, they express
the prodrug-converting enzyme.
• Therefore, when the cells take up the drug, it is converted in to a cytotoxic drug
that kills the tumor.

39.  Anti sense Technology
 RNAi
 Therapeutic ribozymes
 Triple helix forming oligonucleotide gene therapy

41. • ONs- (short) chains of (chemically modified)
ribo- or deoxyribonucleotides.
• Their ability to bind to chromosomal DNA or
mRNA through Watson–Crick and Hoogsteen
base-pairing offers possibilities for highly
specific intervention in gene transcription,
translation, repair, and recombination for
therapeutic applications
• Attacks DNA sequence of a mutated gene to
prevent its transcription
• This therapy uses ssDNA that binds right into
the groove between the double strands of
mutated gene’s DNA.
• The triple helix thus produced , prevents
transcription of DNA.

42.  Biological fluids, like blood serum or
intracellular liquids, contain highly active
nucleases to destroy nucleic acids
 ONs composed of unmodified DNA or RNA
are completely degraded within few hours,
even before they reach their destination
 Solution; Development of modified
nucleotides – higher resistance against
enzymatic degradation-as they are not
regarded as substrates by nucleases
 The 2’-position site for introduction of
functional group, that enhance stability of
ONs
 Phosphorothioates, 2’-O-methoxyethyl
RNA, Phosphorodiamidate morpholino
monomers, PNAs

43.  Silence gene expression and to turn off diseased genes.
 Antisense refers to short DNA or RNA sequences, which are designed to be complementary
to a specific gene sequence.
 During transcription, the sequence of one strand is copied into single strand of mRNA –
sense strand- coding strand-has the code to be read for making the protein. The opposite
strand is called the antisense strand
 Concept of Antisense Technology- is to design an RNA molecule that will serve as
complimentary base pair to mRNA you want to inhibit, thus blocking its translation into protein
 Concept of RNAi; double stranded RNA- molecules are delivered into cells, where the
enzyme Dicer chops them into 21-nt long siRNAs.
 The siRNAs then join with an enzyme complex called RISC, which guides the siRNAs to their
target mRNA, where the bind by complimentary base pairing.
 The RISC complex degrades the si-RNA bound mRNA ,so they cannot bet translated to
proteins.
 Silencing RNA are delivered using plasmid vector, liposomes, Lentiviral delivery, attachment
of siRNAs to cholesterol or fatty acids
 Not possible for multigene diseases

44. Argonaute+
Other Reg.
proteins

46. • Ribozymes are engineered RNA sequences containing enzyme activity capable of
cleaving a specific mRNA sequence.
• Gene therapy can also be used to transfer a gene for a ribozyme into a
defective cell.
The ribozyme has two domains;
 The catalytic domain acts as an enzyme, and
 The recognition domain binds to a specific target RNA.
• Once bound, the enzyme domain cleaves the target RNA.
• Ribozymes may be engineered to recognize and destroy any target mRNA
molecule.
• Transcription of this gene would result in production of the ribozyme RNA. The
ribozyme would then bind and cleave the target mRNA.
• Vector carrying a gene encoding for ribozyme is used for delivery to target cell.

48.  Consists the infusion of engineered T cells that express a Chimeric Antigen
Receptor (CAR) on their cell membrane.
 CAR (Chimeric Antigen Receptor) T cell and gene therapy -manipulates the
patient’s own T cells (immune cells) to attack malignant cancer cells.
 CAR T cancer gene therapy is currently being studied as a treatment for many
different cancer types and has been approved for different forms of leukemia and
lymphoma.
 To be effective, the CAR T cells are engineered to identify and attack only those
cells that have the same antigens your cancer cells have.

49. • The most common procedure for CAR-T cell therapy starts with the extraction of T
cells from the own patient, a process called leukapheresis.
• The T cells are then genetically modified to express a CAR and expanded in vitro.
• Finally, they are reinfused into the patient, ready to fight the tumor.

50. • CAR T cells- genetically engineered to
express a receptor with 2 major functions
 Antigen recognition- through antibody
binding
 T-cell activation-through phosphorylation of
intracellular domains,
CARs consist of three major domains:
• An ectodomain, - extracellular portion -
includes antigen-recognition domain and a
signal peptide.
• Transmembrane domain- supports CAR
stability, and
• An endodomain.; intracellular endodomain
-signal transduction to activate T cells
during antigen recognition
Binding of tumor target antigen -T-cell
activation, proliferation, and target cell
elimination.

52. • Cystic fibrosis: The limitation is that airway epithelial cells are rapidly shed off.
• Severe combined immunodeficiency disease (SCID)
• Growth hormone deficiency: by implanting cultured myoblasts transfected with
GH gene.
• Familial hypercholesterolemia: by introducing LDL receptor gene into
hepatocytes.

53.  The Food and Drug Administration (FDA) has not yet approved any human gene
therapy product for sale.
 Immune response. It reduces gene therapy effectiveness and makes repetitive
rounds of gene therapy useless
 Viral vectors. Immune and inflammatory responses, also fears that viral vector may
recover disease-causing ability
 Target. One does not have control over where the gene will be inserted into the
genome
 Short-lived. Very hard to achieve any long-term benefits
 Multigene disorders. Disorders such as heart disease, Alzheimer's disease,
arthritis, and diabetes, are caused by the combined effects of many genes.

54. What we have learnt..

57.  Gene Therapy of Cancer: Translational Approaches from Preclinical Studies to Clinical
Implementation, edited by Edmund C. Lattime, Stanton L. Gerson Academic Press.
 Therapeutic Oligonucleotides, Jens Kurreck, Royal Society of Chemistry
 Basic and Applied Aspects of Biotechnology, Varsha Gupta, Springer
 Non Viral Vectors in Gene Therapy- An Overview, Murali Ramamoorthy
 Gene therapy: advances, challenges and perspectives , Giulliana Augusta Rangel
Gonçalve, July, 2017
 Nonviral Vectors for Gene Therapy: Lipid- and Polymer-based Gene Transfer Academic
Press
 Biotechnology Fundamentals; Firdos Alam Khan, CRC press
 Biotechnology; Applying the Genetic Revolution, David P. Clark, Nanette J. Pazdernik,
Elsevier Academic Press
 An Introduction to Molecular Biotechnology: Fundamentals, Michael Wink, John Wiley &
Sons
 Polymers and Nanomaterials for Gene Therapy ; Ravin Narain, Woodhead Publishing