Gene Therapy Dr Hanan Hussien Shehata Gene Therapy

Gene Therapy Dr. Hanan Hussien Shehata

Gene Therapy - Strategy A. Gene Replacement Therapy B. Gene Blocking Therapies

Gene Therapy - Strategy A. Gene Replacement Therapy • Loss-of-function mutations (AR). • Replacement of nonfunctional or missing gene product

B. Gene Blocking Therapies • Dominant negative mutations. • Gain-of-function mutations. • Improper regulation mutations.

Dominant negative § § § Some mutations in genes lead to the production of a dominant-negative protein. A dominant-negative protein may block a normal protein from doing its job. In this case, adding a functional copy of the gene won't help, because the dominantnegative protein will still be there causing problems.

Gain-of-function A gain-of-function mutation makes a protein that acts abnormally, causing problems all on its own. For example, let's say a signal activates protein X, which then tells the cell to start growing and dividing. A gain-of-function mutation may make protein X activate cell growth even when there's no signal, leading to cancer.

Improper regulation Sometimes a disorder can involve a protein that is functioning as it should—but there's a problem with where, when, or how much protein is being made. These are problems of gene regulation: genes need to be turned "on" in the right place, at the right time, and to the right level.

Here are some of the newest methods that scientists are developing as potential approaches to gene blocking therapy GENE REPAIRING SMa. RT™ GENE SILENCING Triple-helix-forming oligonucleotides Antisense oligonucleotide……. Ribozymes

GENE REPAIRING SMa. RT™ The term SMa. RT™ stands for "Spliceosome-Mediated RNA Trans-splicing. " This technique targets and repairs the messenger RNA (m. RNA) transcripts copied from the mutated gene. SMa. RT™ works as follows: A complementary RNA strand is generated that pairs specifically with the intron next to the mutated segment of m. RNA. The RNA strand also contains the corrected version of the mutated sequence. When the RNA strand binds to the m. RNA, it prevents the spliceosome from including the mutated segment in the final, spliced RNA product. Since the correct exon is now present, the repaired RNA will now be made into a functional protein

GENE SILENCING Triple-helix-forming oligonucleotides RNA Silencing or inhibition ……. Ribozymes

Gene silencing is an approach used to turn a gene "off" so that no protein is made from it. Gene-silencing approaches to gene therapy can target a gene's DNA directly, or they can target m. RNA transcripts made from the gene.

Triple-helix-forming oligonucleotide gene therapy targets the DNA sequence of a mutated gene to prevent its transcription. This technique delivers short, single-stranded pieces of DNA, called oligonucleotides, that bind specifically in the groove between a gene's two DNA strands. This binding makes a triple-helix structure that blocks the DNA from being transcribed into m. RNA.

A triple-stranded DNA is a structure of DNA in which three oligonucleotides wind around each other and form a triple helix. In this structure, one strand binds to a B-form DNA double helix through Hoogsteen hydrogen bonds. For example, a nucleobase T binds to a Watson– Crick base-pairing of T-A by Hoogsteen hydrogen bonds between an Ax. T pair (x represents a Hoogsteen base pair). An N-3 protonated cytosine, represented as C+, can also form a base-triplet with a C-G pair through the Hoogsteen base-pairing of an Gx. C+. Gx. C

RNA silencing (RNA inhibition) Is a process in which RNA molecules inhibit gene expression typically by causing the destruction of specific m. RNA molecules. This newer method that prevents a particular gene from making protein is called RNA inhibition (RNAi). The aim is to silence the gene by targeting its m. RNA, which carries the genetic message to the protein assembly in the cytoplasm.

Antisense oligonucleotide This technique introduces a short piece of RNA (single stranded) with a nucleotide sequence that is complementary to a portion of a specific gene's m. RNA transcript. The short piece of RNA will find attach to its complementary sequence, forming a double-stranded RNA molecule, which the cell then destroys

Antisense oligonucleotide The majority of the antisense drugs investigated in the clinic function via an RNase H-dependent mechanism. RNase H is a ubiquitous enzyme that hydrolyzes the RNA strand of an RNA/DNA duplex. Oligonucleotide-assisted RNase H-dependent reduction of targeted RNA expression can be quite efficient, reaching 80– 95% down-regulation of protein and m. RNA expression.

Antisense oligonucleotide

RNA interference (RNAi) Two types of small (RNA) molecules micro. RNA (mi. RNA) and small interfering RNA (si. RNA) – are central to RNA interference. These small RNAs can bind to other specific messenger RNA (m. RNA) molecules and prevent them from producing a protein.

Types of small (RNA) molecules Short interfering RNAs Researchers design short RNAs (called short interfering RNAs, or si. RNAs) that will prompt a cell to destroy matching RNA, preventing the production of a protein from a particular gene. micro. RNAs Class of small non-protein coding RNA molecules, 19– 22 nts. They negatively regulate gene expression at the post-transcriptional level

RNA interference (RNAi) biogenesis A certain protein called Dicer cleaves the double stranded RNA (dsi. RNA) and pre-micro. RNA (pre-mi. RNA) into short double-stranded RNA fragments called small interfering RNA and micro. RNA respectively. These fragments are approximately 20 -25 base pairs long. Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. RISC has a catalytic component , which is an endonuclease capable of degrading messenger RNA (m. RNA).

MECHANISM OF DOWN REGUATION Down regulation of gene expression occur by either of two posttranscriptional mechanisms: m. RNA cleavage, or translational repression. The choice is determined by the degree of complementarity between the small interfering RNA or micro. RNA and their target m. RNAs. The perfect complementarity triggers the action of Slicer an endonuclease, which acts through cleaving the target m. RNA. Central mismatches excludes cleavage and promotes translational repression of m. RNA target

Employment of Exosomes in gene therapy Exosomes are 50– 90 nm diameter vesicles, comprising a lipid bilayer and enriched nucleic acid and protein content. Released in Extracellular medium by a broad array of cells They are crucial messengers that can regulate cellular processes by ‘trafficking’ molecules from cells of one tissue to another Exosomes carry protein, lipids, m. RNA & noncoding RNA

Delivering nc. RNA mimics or inhibitors to target cells via exosomes The combination of exosomes with RNAi technology is a promising method for gene therapy. Exosomes transfer exogenous si. RNAs to recipient cells as monocytes, lymphocytes, liver cells……. which will knock down the target gene in those recipient cells Exosomes derived from various cell types have been also used to deliver tumor suppressor mi. RNAs (mi. Rs mimics) or oncogenic mi. RNA inhibitors (anti-mi. Rs), to recipient cells both in vitro and in vivo. By manipulating expression of exosomal surface markers, organ specific delivery may also be achieved

Ribozyme gene therapy Ribozyme gene therapy targets the m. RNA transcripts copied from the gene. Ribozymes are RNA molecules that act as enzymes. Most often, they act as molecular scissors that cut RNA. In ribozyme gene therapy, ribozymes are designed to find and destroy m. RNA encoded by the mutated gene so that no protein can be made from it.

Ribozymes Genetic lesions could be targeted therapeutically through ribozymes, provided that the sequence of the genetic information involved in the disease is known. The hammerhead ribozyme, one of the smallest ribozymes identified, is able to induce site-specific cleavage of RNA.

Hammerhead ribozyme has the ability to down-regulate gene expression through RNA cleavage makes the hammerhead ribozyme a candidate for genetic therapy. The ribozyme and the substrate are two different oligo ribonucleotides with only some regions of complementarity.

Hammerhead ribozyme The trans-acting ribozymes consist of a catalytic core, flanked by two arms that share complementarity with the RNA sequence to be cleaved. The Watson–Crick base pairing between ribozyme and substrate, forming helices I and III, makes the cleavage of target RNA molecules sequence specific

Hammerhead ribozyme Site of cleavage

Genetically modifying immune cells As part of its natural function, the immune system makes large numbers of white blood cells, each of which recognizes a particular molecule (or antigen) that represents a threat to the body. Researchers isolate an individual's immune cells and genetically engineer them through gene therapy to recognize a specific antigen, such as a protein on the surface of a cancer cell or an abnormal protein. When returned to the patient, these modified cells will find and destroy any cells that carry the antigen.

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