Gene Therapy- Types, Methods, Vectors, Major Development, Problems

Sanju Kumari

• 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

Types of gene therapy

Germ line gene therapy Somatic cell gene therapy

Somatic cell gene therapy

IN VIVO EX VIVOEX VIVO

Hematopoetic stem cell gene therapy

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

Vectors for gene therapy

• Viral vectors Retroviral vectors Lentiviral vectors Adeno associated viral

vectors Adenoviral Vector

• Non-Viral vectors Oligonucleotides Naked-DNA Polyplexes Lipoplexes Liposomes

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

Retroviral life cycle

• 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

Production of Retroviral particle

(Verma & Weitzman, 2005)

• 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

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

Adenovirus

Adenoviral-mediated Rybp expression promotes tumorcell-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

Ad-Rybp infection induces apoptosis

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

• 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

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

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

Lipoplexes and polyplexes mediated transfection

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

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

Methods of gene transfer

• Physical methods :- Microinjection Electroporation

• Chemical methods:- Calcium phosphate precipitation

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.

Optical damage of astrocyte irradiated by femtosecond laser

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)

Calcium phosphate transfection

• Most popular tools in neuroscience research • Low cell toxicity and easiness to use

Fluorescent images of GFP-transfected cells

Chimeraplast

(Richardson et.al.,2002)

Triplex forming oligonuleotide

(Richardson et.al.,2002)

Small fragment homologus replacement

(Richardson et.al.,2002)

• 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)

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)

• 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)

Vectors for SleepingBeauty-mediated transposition

Genetic assay fortransgene integration in cultured cells.

Genetic assay used to recover transposons from mouse chromosomes

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)

Hair follicle formation in mice skin treated with MEND3

The multifunctional envelope type nano device

Major Development

First Approved Gene Therapy First Approved Gene Therapy

Ashanthi De Silva - A rare genetic Ashanthi De Silva - A rare genetic disease called severe combined disease called severe combined immunodeficiency (SCID)immunodeficiency (SCID)

Defective adenosine deaminase Defective adenosine deaminase gene results in deficiency of ADA gene results in deficiency of ADA protein protein

It plays important role in It plays important role in deamination reactiondeamination reaction

Lack of healthy immune systemLack of healthy immune system Dr. W. French Anderson Dr. W. French Anderson with four-year old Ashanthi with four-year old Ashanthi De Silva at U.S. National De Silva at U.S. National Institutes of HealthInstitutes of Health

• 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)

Gene correction using retroviral vectors

No. of cells after gene No. of cells after gene transfertransfer

Normal sized thymusNormal sized thymus

Repeat administration of DNA / liposomes to the nasalepithelium 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)

CFTR immunohistochemical staining of nasal brushing cells

Third strand-mediated psoralen-induced correctionof the sickle cell mutation

• Plasmid containing a b-globin gene fragment transfected into cells

Interaction of the psoralen delivery strand with the target sequence

Screening assay for mutation correction

(Varganov (Varganov et alet al., 2007)., 2007)

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)

Human htt expression by Human htt expression by RNAiRNAi

RNAi expression to RNAi expression to mouse striatummouse striatum

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

Correction of aberrant splicing by U7.623 snRNA in -globin IVS2 mutant cells

(Vacek (Vacek et al., et al., 2003)2003)

Dystrophin expression in mice by intravascular injection of naked DNA

Duchenne muscular dystrophy (DMD) - Duchenne muscular dystrophy (DMD) - lethal, X-linked,recessive disease caused by a defect in the dystrophin gene

Injection of DNA solution by tail Injection of DNA solution by tail artery and tail veinartery and tail vein

Effect of histamine on gene Effect of histamine on gene expressionexpression

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)

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 (Pardridge et al.,et al.,2005)2005)

• 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)

First Deafness Cure in guinea pig

• 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

Gene Therapy for Advanced Melanoma

• 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

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

Results

Assesment of visual mobilityAssesment of visual mobility

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

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

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 toxicityUniversity 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