GENE THERAPYGENE THERAPY

M YOUSRY ABDEL-MAWLA,MDM YOUSRY ABDEL-MAWLA,MD

ZAGAZIG FACULTY of ZAGAZIG FACULTY of MEDICINE,EGYPTMEDICINE,EGYPT

DNA replicationDNA replication

• The DNA in the chromosomes is The DNA in the chromosomes is replicated during a period of replicated during a period of interphase called S-phase of cell interphase called S-phase of cell cycle which stands for synthesis of cycle which stands for synthesis of DNA.DNA.

• strands could come apart and each strands could come apart and each separated strand serve as separated strand serve as template for the synthesis of a template for the synthesis of a new partner strand new partner strand complementary in nucleotide complementary in nucleotide sequence.sequence.

DNA repair systemDNA repair system::

• Mismatch repairMismatch repair : a pair of non-hydrogen : a pair of non-hydrogen bonded bases (e.g. G-----T) within a helix is bonded bases (e.g. G-----T) within a helix is recognized as aberrant and a polynucleotide recognized as aberrant and a polynucleotide segment of daughter strand is excised, segment of daughter strand is excised, thereby removing one member of the thereby removing one member of the unmatched pair. unmatched pair.

• Nucleotide excision repairNucleotide excision repair : lesions that : lesions that distort the double helix as a thymine dimer distort the double helix as a thymine dimer can also be repaired by the excision of a can also be repaired by the excision of a short stretch of nucleotides including the short stretch of nucleotides including the lesion, followed by its correct replacement , lesion, followed by its correct replacement , the opposite strand serving as the templatethe opposite strand serving as the template. .

DNA replicationDNA replication

• Base excision repair in which Base excision repair in which deamination converts cytosine to deamination converts cytosine to uracil and adenine to hypoxanthin. uracil and adenine to hypoxanthin.

• DNA glycosylases recognize the DNA glycosylases recognize the abnormal bases and hydrolyse them abnormal bases and hydrolyse them of, leaving apurin or apyrimidine of, leaving apurin or apyrimidine sites in which the deoxyribose has no sites in which the deoxyribose has no base attached to it..base attached to it..

Gene therapyGene therapy

• Gene therapy is divided into : Gene therapy is divided into : • Germ line gene therapyGerm line gene therapy• Somatic gene therapy.Somatic gene therapy.• Germ line gene therapy: the therapeutic gene Germ line gene therapy: the therapeutic gene

modification is introduced into all cells of the modification is introduced into all cells of the body or a subset of cells including germ cells.body or a subset of cells including germ cells.

• Somatic gene therapy: theSomatic gene therapy: the genetic genetic modificationmodification is restricted exclusively to is restricted exclusively to somatic cells with no effect on the germ line. somatic cells with no effect on the germ line.

Gene DeliveryGene Delivery::

• In vivo delivery:In vivo delivery: direct introduction of direct introduction of genetic material into the skin of the patient genetic material into the skin of the patient

Treatment of metastatic malignant Treatment of metastatic malignant melanoma. Skin tumors were injected melanoma. Skin tumors were injected directly with plasmid DNA designed to directly with plasmid DNA designed to express the human leucocyte antigen express the human leucocyte antigen (HLA) class I gene, B7, which is chosen to (HLA) class I gene, B7, which is chosen to be mismatched with the patient's HLA type be mismatched with the patient's HLA type 9.9. In vivo gene therapy may more In vivo gene therapy may more accurately represent the actual accurately represent the actual interactions between the skin and interactions between the skin and surrounding tissuessurrounding tissues

Ideal in vivo gene delivery Ideal in vivo gene delivery systemsystem

• High efficiency of uptake of the High efficiency of uptake of the therapeutic gene by the target therapeutic gene by the target cells, transportation of the cells, transportation of the therapeutic gene to the nucleus of therapeutic gene to the nucleus of the target cell with minimal of the target cell with minimal of intracellular degradation and intracellular degradation and sustained expression of the sustained expression of the therapeutic gene at a level that therapeutic gene at a level that alleviates the conditionalleviates the condition

Ex vivo deliveryEx vivo delivery

• This involves removal of a skin sample from This involves removal of a skin sample from the patient, followed by propagation of skin the patient, followed by propagation of skin cells (eg stem cells)in culture, introduction cells (eg stem cells)in culture, introduction of genetic material into the cultured cells, of genetic material into the cultured cells, and return of the epithelailized genetically and return of the epithelailized genetically engineered cells in the form of a skin graft engineered cells in the form of a skin graft back to the patient .Ex ex vivogene therapy back to the patient .Ex ex vivogene therapy using stem cell in treatening of using stem cell in treatening of epidermolysis bullosaepidermolysis bullosa

Gene delivery systemsGene delivery systems::

• Gene delivery systems include viral and non Gene delivery systems include viral and non viral vectors viral vectors

• The ideal vector as a mean of delivering The ideal vector as a mean of delivering genes to human cells and tissues is that genes to human cells and tissues is that vector which delivers genes with high vector which delivers genes with high efficiency into theefficiency into the proper tissue.proper tissue.

• Ideal vector should either remain in a stable Ideal vector should either remain in a stable extra-chromosomal state or to have the extra-chromosomal state or to have the ability to target a specific site within the ability to target a specific site within the genomegenome. .

Biologic viral vectors.

Type of vectorsIntegrationTo gemone

AdvantagesLimitations

- Retrovirus-Molony murine leukaemia Virus

Yes-Wide host range; high efficiency; transduction of dividing cells; efficient expression of foreign gene product; stable

integration; infects only once and does not replicate in vivo.

-Does not infect non dividing terminally differentiated cells, may be oncogenic optimal insert size 5-7 kb.

-Leintvirus (HIV) Yes-Wide host range; stable transduction of dividing and non dividing terminally differentiated cells with long term expression; nonpathogenic; lack of expression of viral

proteins.

-AdenovirusNo-Transduction of non dividing cells with high efficiency; wide host rang; high viral titer and high expression levels; newly developed gutless vectors have insert size as large as 30 kb.

-Expression of viral proteins results in toxic reaction and inflammation; carcinogenic; low efficiency in dividing cells,

short-term expression, insert size only 7-11 kb.

-Adeno-associated virusYes-Transduction of dividing and non dividing cells, all viral coding sequences can be deleted except those required for transduction; non immunogenic and nonpathogenic; long-

term expression of transgene; specific integration site (some forms).

-Limited transduction efficiency that depends on helper viral functions, although in newer systems helper virus not needed, low efficiency of integration to genome; small insert size~4-5

kb.

-Herpes simplex virusNo- Transduction of neurons and glial cells, wide host range; large insert size up to 30 kb; efficient infection.

-Short-term expression; spreading of the infection to surrounding cell populations; new engineered vectors are

avirulent in surrounding terminally differentiated cells; immunogenic.

Non viral vectors.

Vector systemAdvantagesDisadvantages

1-Nacked plasmid DNA - simple, relatively efficient, non immunogenic, no mutagenesis.

- Transient gene expression, DNA not integrated into the genome, remain episomal.

2- Chemical vectorsa-Calcium phosphate b-Cationic liposomes(lipoplex)c-Polylysine-DNA complexes

- Easy to use.- Non infectious, non immunogenic, effective for in vivo gene transfer, can carry large DNA fragments.- Targeted delivery, can carry large fragments of DNA.

- Random integration, inefficient DNA transfer.-- -- Unstable, remain episomal, poor gene expression. -Unstable, remain episomal, poor or gene expression.

3- Physical methodsa- Electroporation

b-Gene gun approa-chc-Microneedle injection

- Easy to use, safe, many cell type and different applications.- Cell receptor independent, delivers genes to different tissues. - used in vaccine protocols, effective in localized area, increase gene expression, painless, no bleeding.

--- Random integration unless targeted, inefficient

DNA transfer, Superficial burn.

-No integration of DNA, transient gene expression.

- Ineffective in large surface area.

4-Biologic non viral vectors human artificial chromosomes

- Stable, non-infectious, can carry large fragments of DNA, non immunogenic, no

integration into the genome.

- Still in developmental stages.

Applications of gene Applications of gene therapytherapy• 1-Gene therapy for systemic diseases via the 1-Gene therapy for systemic diseases via the

skin: skin:

a- Skin acts as a secretory organ e.g. in a- Skin acts as a secretory organ e.g. in haemophilia and growth hormone haemophilia and growth hormone deficiency.deficiency.

b- Skin acts as a metabolic sink or a b- Skin acts as a metabolic sink or a bioreactor in the following metabolic disorders bioreactor in the following metabolic disorders e.g. - Adenine deaminase e.g. - Adenine deaminase deficiency ,Ornithine aminotransferase deficiency ,Ornithine aminotransferase deficiency or hypercholestrelemia.deficiency or hypercholestrelemia.

Applications of gene therapyApplications of gene therapy• 2- 2- Inhereted skin diseases e.g. xeroderma Inhereted skin diseases e.g. xeroderma

pigmentosum, ichthyoses (X-linked and lamellar pigmentosum, ichthyoses (X-linked and lamellar types), epidermolysis bullosa (junctional and types), epidermolysis bullosa (junctional and dystrophic types).dystrophic types).

• 3-3- Skin malignancies e.g. malignant metastatic Skin malignancies e.g. malignant metastatic melanoma and cutaneous T-cell lymphoma.melanoma and cutaneous T-cell lymphoma.

• 4-4- Congenital hair disorders. Congenital hair disorders.• 5-5- Wound healing.. Wound healing..• 6-6- DNA vaccine. DNA vaccine.• 7-7- Genetic pharmacology. Genetic pharmacology.• 88 Pro-drug activation or suicide gene for cancer. Pro-drug activation or suicide gene for cancer.• 9 - 9 - Nucleic acid agents include antisense Nucleic acid agents include antisense

technology technology (RNA and oligonucleotides), ribozymes, and (RNA and oligonucleotides), ribozymes, and

splicisome-mediated RNA trans-splicing.splicisome-mediated RNA trans-splicing.• 10- 10- Nucleic acid pharmaceuticals.Nucleic acid pharmaceuticals.

Antisense Gene Therapy (AS-ODNs)

Antisense Gene Therapy (AS-ODNs)

•The discovery of antisense oligonucleotides (AS-ODNs) and small interfering RNA( siRNA) has opened wide perspectives in therapeutics for the treatment of cancer, infectious and inflammatory diseases or to block cell proliferation and diseases caused thereby.

Antisense therapyAntisense therapy

Antisense therapyAntisense therapy is a form of is a form of treatment for genetic disorders or treatment for genetic disorders or infections. When the genetic sequence of infections. When the genetic sequence of a particular gene is known to be causative a particular gene is known to be causative of a particular disease, it is possible to of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) bind to the messenger RNA (mRNA) produced by that gene and inactivate it, produced by that gene and inactivate it, effectively turning that gene "off". effectively turning that gene "off".

Antisense TherapyAntisense Therapy

Antisense therapy Antisense therapy is not strictly a is not strictly a form of gene form of gene therapy, but is a therapy, but is a genetically-genetically-mediated therapy mediated therapy and is often and is often considered considered together with together with other methodsother methods

Small interfering RNA ( siRNA)

•Gene expression could be inhibited by the introduction of double-stranded RNA with sequence complementarity to the gene being targeted, a mechanism that was named RNA interference ( siRNA)

Short interfering RNA(siRNA)• long, double-stranded RNAs are

introduced into a cell, they become diced into short, double-stranded, 21-nt RNAs containing 2-nt 39 overhangs, known as

short interfering RNA (siRNA).

• The siRNA then guide cellular machinery to target and degrade mRNA with a similar sequence.

Antisense oligonucleotoids(AS-ODNs)

• Synthetic single-stranded DNA fragments that bind to specific intracellular messenger RNA strands (mRNA) forming a short double helix. They consist of short sequences, composed of 13 to about 25 nucleotides*, which are complementary to mRNA strands in a region of a sequence designed as sense strand.

• By binding to the mRNA molecules, AS-ODNs are shown to stop translation of the mRNA, and hence protein synthesis expressed by the targeted gene.

Methods for overcoming the skinbarrier against gene delivery• To achieve more efficient cutaneous gene

delivery,removal of the horny layer is thought to be the best way to disrupt the barrier of the skin. Tape-stripping using adhesive tape may be used to remove the horny layer.

• Several technological advances have been made in overcoming this barrier: electroporation, sonophoresis iontophoresis and chemical penetration enhancers (CPEs).

MicroRNAs and the skin• Humans inherit 23 chromosomes from

each parent to form a diploid genome consisting of 46 chromosomes.

• The majority of the genome actually consists of non-coding genes and regions. For a long time

• The majority of the DNA in our genomes, initially labeled as unnecessary

• Useless DNA, is actively transcribed into functional primary RNA ,it transcripts or non-coding RNAs (ncRNA)

Non-coding RNAs (ncRNA)

Few ncRNAs are characterized: Ribosomal RNAs (rRNAs)

Transfer RNAs (tRNAs)

• MicroRNAs are small 21–25 nt RNA molecules that are essential regulators of a wide range of cellular processes

Micro RNAs(MiRNAs)vs siRNAs

• MiRNAs refer to small RNAs produced naturally from the human genome, and have diverse and widespread roles.

• They are generated by transcribing a single RNA

• siRNAs can be either exogenous or endogenous—that is, either naturally occurring in the genome or introduced from outside the cell.

MicroRNAs (miRNAs) Functions

•MicroRNAs (miRNAs) are very small endogenous RNAmolecules about 22–25 nucleotides in length, capable of post-transcriptional gene regulation.

• miRNAs bind to their target messenger RNAs (mRNAs), leading to cleavage or suppression of target mRNA translation

The Biogenesis of miRNAs

• miRNAs are transcribed by RNA polymerase II in mammalian cells

• The primary miRNA transcript (pri-miRNA) is usually several kilobases long, poladenylated at its 3* end and capped with a 7- methylguanosine cap at its 5*end .

• The intranuclear RNase III enzyme then cleaves the pri-miRNA, which may contain multiple miRNA, into several precursor miRNAs (premiRNAs). DGCR8 (DiGeorge syndrome critical region gene 8/) is essential for RNASE activity

Molecular biologyMolecular biology

• Heritable genetic information is Heritable genetic information is contained in DNA, which can be contained in DNA, which can be replicated and passed to daughter replicated and passed to daughter cells. cells.

• DNA is transcribed to RNA, transported DNA is transcribed to RNA, transported to the nucleus, and translated into to the nucleus, and translated into proteins. The identification of reverse proteins. The identification of reverse transcriptase demonstrated that RNA transcriptase demonstrated that RNA can also be converted back into DNAcan also be converted back into DNA. .

Molecular biologyMolecular biology

• Gene activity is regulated on many Gene activity is regulated on many levels. Representative mechanisms levels. Representative mechanisms of gene regulation are shown at of gene regulation are shown at the DNA, RNA, and protein levels. the DNA, RNA, and protein levels. The rate of gene transcription can The rate of gene transcription can be affected by the quantity of be affected by the quantity of transcription factors (green transcription factors (green circles) that are locally available to circles) that are locally available to interact with the gene interact with the gene

Molecular biologyMolecular biology• DNA is packaged among histone proteins DNA is packaged among histone proteins

(spheres), which can be modified (red (spheres), which can be modified (red octagons) in a way to package DNA more octagons) in a way to package DNA more tightly and make it less accessible to tightly and make it less accessible to transcription factors. transcription factors.

• On the RNA level, the stability of a On the RNA level, the stability of a transcript can determine how long it transcript can determine how long it persists in the cell and how much protein persists in the cell and how much protein can be made. At the protein level, proteins can be made. At the protein level, proteins can be switched to active form by chemical can be switched to active form by chemical modifications, such as phosphorylation modifications, such as phosphorylation (gold star) or targeted for destruction by (gold star) or targeted for destruction by ubiquitination (pink hexagons). ubiquitination (pink hexagons). Polyubiquitination causes proteins to be Polyubiquitination causes proteins to be ferried to the proteasome, which degrades ferried to the proteasome, which degrades proteins into short amino acids. proteins into short amino acids.

• MicroRNAs function at the level of altering MicroRNAs function at the level of altering RNA stability, as well as by affecting the RNA stability, as well as by affecting the rate at which RNAs are translated into rate at which RNAs are translated into proteinsproteins

MicroRNAs in Cutaneous Biology.

MicroRNAs (miRNAs) and MicroRNAs (miRNAs) and short interfering RNAs short interfering RNAs (siRNAs)(siRNAs)

• They are classes of regulatory small RNA They are classes of regulatory small RNA molecules, ranging from 18 to 24 molecules, ranging from 18 to 24 nucleotides in length,nucleotides in length,

• Their roles in development and disease Their roles in development and disease are becoming increasingly recognized. are becoming increasingly recognized.

• They function by altering the stability or They function by altering the stability or translational efficiency of messenger RNAs translational efficiency of messenger RNAs (mRNAs) with which they share sequence (mRNAs) with which they share sequence complementarity, and are predicted to complementarity, and are predicted to affect up to onethird of all human genes. affect up to onethird of all human genes.

miRNAs and psoriasis• Tumor necrosis factor (TNF)-a is a proinflammatory

cytokine shown to play an important role in the pathogenesis of psoriasis

• Three different miRNAs have thus far been associated with this skin disease and linked to both innate immune responses and the TNF-a pathway

• miR-203 was the first miRNA found to be significantly overexpressed in psoriasis patients

• Up-regulation of miR-203 leads to down-regulation of suppressor of cytokine signaling-3 (SOCS-3) expression in psoriatic skin

• miR-146a is overexpressed in many psoriatic skin lesions and patients with rheumatoid arthritis

• In contrast, miR-125b is down-regulated in psoriasis

•SOCS-3 is an,inhibitor of the signal transducer and activator of transcription 3 (STAT3) pathway , which is widely expressed and activated by various growth-regulating signals and inflammatory cytokines such as interleukin-6 or interferon-

•STAT3 plays a critical role in many biological activities, such as cell proliferation, migration, homeostasis, inflammation, immune regulation and oncogenesis

SOCS-3 Vs STAT3

•STAT3 has been shown to be constitutively activated in epidermal keratinocytes of human psoriatic lesions

• Inhibition of STAT3 has drastically improved clinical prognoses in psoriatic patients

miR-146a and psoriasis

•miR-146a targets, TNF receptor-associated factor 6(TRAF6) and IL-1R-associated kinase (IRAK) which are all involved in the TNF-a pathway, which contributes to psoriatic skin inflammation.

miRNAs and wound healing•Wound healing can be divided into

four phases: inflammatory, proliferative, fibroplasia maturation, and remodeling phase.

•Platelets secrete various cytokines, including platelet-derived growth factor (PDGF), platelet factor IV and transforming growth factor beta

(TGF-b)•miR-140 has been shown to have a

modulating effect on PDGF receptor a.

miRNAs and wound healing• Polymorphonuclear leukocytes and

macrophages migrate to the wound site and release a variety of chemotactic factors such as fibroblast growth factor (FGF), TGF-band TGF-a, plasma-activated complements C3a and C5a, interleukin-

1 (IL-1), tumor necrosis factor (TNF) and PDGF.

• TNF-a is regulated by miR-146a targets

miRNAs and angiogenesis•The role of miRNAs in angiogenesis

has been the subject of numerous studies

•overexpression of miR-221 and miR-222 indirectly reduces the expression of endothelial nitric oxide synthase (eNOS), which is essential for many endothelial cell functions

miRNAs and skin cancer

• miRNAs and their key regulators are essential for morphogenesis of the skin

and hair follicles. • It is thus expected that a disruption

of miRNA expression can be observed in

various malignant skin lesions. • miR-218-1 is a tumor suppressor

inactivated in breast, lung and colorectal cancers. It is located within the tumor suppressor gene SLIT2 (human homologue of Drosophila Slit2)

miR and Melanoma

• miR-137 modulates expression of microphthalmia-associated transcription factor (MITF), which is a major regulator of melanocyte growth, maturation, apoptosis and pigmentation

• miR-221 & miR-222,indirectly regulate MITF expression

• Ultraviolet radiation-induced sun tanning occurs through keratinocyte expression of a-melanocyte stimulating hormone (a-MSH), which then leads to melanocyte MITF expression.

• MITF induction protects the skin from DNA damage.

• Expression of melanoma inhibitor of apoptosis (MLIAP) in melanoma cells is MITF-dependent

miR-221 & miR-222 in Melanoma

•miR-221 & miR-222 primarily control melanoma progression through down-regulation of cyclin-dependent kinase inhibitor 1b (p27Kip1/CDKN1B) and c-KIT receptor, both of which play critical roles in melanocyte physiology and favor induction of malignant phenotypes

miR &Kaposi’s sarcoma• A form of skin cancer associated with herpesvirus (KSHV), has been identified as a

causative agent of several diseases such as primary effusion lymphoma (PEL).

• Human miR-155 shares several targets and binding sites such as the transcriptional regulators BACH-1, FOS and the proapoptotic

effector LDOC-1 with viral miR-k12-11.• The possibility that mir-k12- 11 may play a

role in tumorgenesis by interfering in the network of transcripts that are regulated by miR155 indicates a possible link between viral and non-viral tumorigenesis

•TNF-a signaling has been closely tied with tumor formation, and its activation upregulates the nuclear transcription factor nuclear factor kappa B (NF-kb).

•NF-kb is broadly involved with inflammatory responses, immunity, and protection against apoptosis.

•Cylindromatosis tumor suppressor gene, CYLD, is a suppressor of NF-kb activation

•CYLD functions as a deubiquitinating enzyme, responsible for removing ubiquitin groups from specific proteins.

•Ubiquitination of TNF-receptor related factor (TRAF ) activates its association with the inhibitor of kappa-beta kinase complex (Ikb), leading to upregulation of NF-kb and prevention of apoptosis

• In a normal state, CYLD functions to block TRAF ubiquitination, and protects against NF-kb activation.