Molecular Technology: Lecture 3

genetic engineering

the deliberate modification of the characteristics of an organism by manipulating its genetic material

gene therapy

• a normal gene is inserted to compensate for a nonfunctional gene and an abnormal gene can be repaired through selective reverse mutation

• genetic diseases like cystic fibrosis, blood disorders, muscular dystrophy, etc.

• can be applied as therapy for cancers, inherited disorders, infectious diseases, and immune system disorders

gene therapy technique

correcting defective genes that are responsible for disease development

gene therapy application

genetic principles in the treatment of human disease to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized to improve a genetic disorder

gene therapy production

production of genetic material into normal cells in order to conteract the effect of a disease gene o introduce a new function

effective approaches of gene therapy

1. gene transplantation: a normal gene inserted to compensate for a nonfunctional gene

2. gene correction: an abnormal gene repaired through selective reverse mutation

3. gene augmentation: change the regulation of gene pairs

gene transfer

• restriction enzymes cut the desired gene from the genome and into the plasmid

• since it is the same enzyme the bases are left with sticky ends

• ligase joins the sticky ends

• the recombinant plasmid is inserted into the host cell

target cells to gene transfer

• in vitro targeting healthy and cancerous cells

• scientists can target cancer cells with genes that can be used to destroy the cells “suicide genes"

somatic cell gene therapy

• theraputic genes transferred into the somatic cells

• will NOT be inherited in later generations

• all researches directed to correct genetic defects in somatic cells

germ line gene therapy

• therapeutic genes transferred into the germ cells

• transfer of a section of DNA to cells that produce eggs ot sperm

• it is heritable and passed on to later generations

• not being attempted due to ethical, safety, and technical reasons

strategies for transgene delivery

In vitro:

• cells removed from body —> transgene delivered cells cultured —> cells returned to the body

In Vivo

• transgene delivered directly into host

ex vivo gene therapy

1. isolate cells with genetic defect from a patient

2. grow the cells in culture

3. introduce the theraputic genes

4. select genetically corrected cells and grow them

5. transplant the modified cells to the patient

In vivo gene therapy

• direct delivery of theraputic gene into target cell into patients body

• carried out by viral or non viral vector systems

• it can be the only possible option in patients where individual cells cannot be cultured in vitro in sufiicient numbers

• necessary when cultured cells cannot be reimplanted in patients effectively

requirements of vectors

• not be identified by the immune system

• stable and easy to reproduce

• have longevity

• high efficacy

• high specificity and low toxicity

• should be able to protect and deliver DNA across the cell membrane into the nucleus

• should be easy to be produced in large amounts and be inexpensive

• target gene delivery to specific cells

retrovirus vector system

• the recombinant retroviruses have the ability to integrate into the host genome in a stable fashion

• can carry a DNA less than 3.4 kb

• replication defective virus particles

• target cell dividing

adeno virus vector system

• adeno virus with a DNA genome (good vectors)

• target non dividing human cell

adeno associated virus vector

• it is a human virus that can integrate into chromosome 19

• single stranded, non pathogenic small DNA virus

• AAV enters host cell, becomes double stranded and gets integrated into chromosome

herpex simplex virus vector

• viruses which have natural tendency to infect a particular type of cell

• infect and persist in nervous cells

pure DNA construct

• direct introduction of pure DNA construct into the target tissue

• the efficiency of DNA uptake by cells and expression rather low

• consequently, large quantities of DNA have to be injected periodically

lipoplexes

• lipid DNA complexes; DNA construct surrounded by artificial lipid layer

• most of it gets degraded by lysosomes

gene gun

employs a high pressure delivery system to shoot tissue with gold or tungsten particles that are coated with DNA

microinjection

• process of using a glass micropipette to insert microscopic substances into a single living cell

• normally performed under a specialized optical microscope setup called a micromanipulator

using detergent mixtures

• certain charged chemical compounds like calcium phosphates are mixed with functional cDNA of desired function

• the mixture is introduced near the vicinity of recipient cells

• the chemicals disturbs the cell membrane, widens the pore size and allows cDNA to pass through the cell

Lipofection

• a technique used to inject genetic materials into a cell by liposomes

• liposomes: artificial phospholipid vesicles used to deliver a variety of molecules including DNA into the cells

gene augmentation therapy

• most common form of gene therapy

• foreign gene replaces missing or defective gene

• adds DNA containing a functional version of the lost gene back into the cell, the new gene produces a functioning product at sufficient levels to replace the protein that was missing

gene inhibition therapy

• done to block the overproduction of some proteins by introducing a gene whose product is either inhibiting the expression of another gene or interferes with the activity of the product of another gene

• antigene: blocks transcription using antigene oligonucleotide

• antisense: blocks translation using antisense oligonucleotide

killing specific cells

• insert DNA into a diseased cell which leads to cell death

• inserted DNA contains a “suicide” gene that produces a highly toxic product, which kills the diseased cell

• OR inserted DNA causes expression of a protein that marks the cells so that the diseased cells are attacked by the body’s natural immune system

gene therapy advantages

• potential to eliminate and prevent hereditary diseases such as cystic fibrosis

• it is a possible cure for heart disease, AIDS and cancer

• gives someone born with a genetic disease a chance to life

• it can be used to eradicate diseases from the future generations

gene therapy disadvantages

• long lasting therapy is not achieved by gene therapy; due to rapid dividing of cells benefits of gene therapy is short lived

• immune response to the transferred gene stimulates a potential risk to gene therapy

• viruses used as vectors for gene transfer may cause toxicity, immune responses, and inflammatory reactions in the host

• disorders caused by defects in multiple genes cannot be treated effectively using gene therapy

BMP’s

• bone morphogenic proteins enable skeletal tissue formation during embryogenesis, growth, adulthood, and healing

• BMPs 2, 4, and 7, are the only growth factors that singly induce de novo bone formation in both in vitro and at heterotrophic sites

bone repair gene therapy

regeneration of the bone structure in treatment of bone anomalies, tooth loss, TMJ diseases, traumatic amputations, and consequences of tumor resection

PGDF

delivery of platelet derived growth factor by gene transfer has been shown to stimulate gingival fibroblast AND positive effects in regenerating bone around teeth and dental implants

BSP

bone sialoprotein is a major non collagenous protein in bone and other mineralized tissues

DNA vaccination

• the ability to induce an immune response to a protein antigen by administration of plasmid DNA encoding the antigen has been successfully demonstrated in animal models

• immunization of salivary gland using plasmid DNA encoding the P gingivalis fimbrial gene leads to the production of fimbrial protein locally in the salivary gland tissue

oral cancer

• generally demonstrate impaired cell-cycle progression, largely due to mutations and teh over expression of cell-cycle regulators

• general strategy in cancer treatment is to express a gene product that will result in cancer cell death

gene therapy to grow new teeth

• more than 200 genes are expressed during tooth development

• PAX 9: master gene

• RUNX2 or USAG1: repress or activate genes used to stimulate the third dentition in order to induce new tooth formation in mice

CRISPR-Cas9

a genome editing tool that is faster, cheaper and more accurate than other DNA editing techniques

• edit parts of the genome by removing, adding, or altering sections of the DNA sequence

• cas9 nuclease stays the same regardless of target DNA

• changing 20-21 nucleotides in the gRNA alters the sequence specificity of the CRISPR-cas9 complex

• can make a large library

• mixing Cas9 with more than one guide RNA allows for multiplexing (targeting many sites at once)

gRNA

guide RNA

crRNA

CRISPR RNA is produced from spacer DNA of CRISPR system which has the complementary sequence to target DNA

tracrRNA

trans-activating crRNA, small trans-encoded RNA which helps in maturation of crRNA

Cas9

• RNA-guided DNA endonuclease found in streptococcus pyogenes and other bacteria

• unzips the dsDNA as well as acts as a pair of molecular scissors that can cut the two strands of DNA at a specific location in the genome so bits of DNA can ten be added or removed

Target DNA

the DNA molecule which has to be cleaved or edited which is basically a viral genome entered into the bacteria

PAM

• protospacer adjacent motif

• 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR system

• essential targeting component which distinguishes bacterial self from non self

cas9 mechanism

1. infection by virus, entry of viral genome into the bacterial cell

2. formation of cas9 complex

• cas genes produces cas9 nuclease

• crRNA is transcribed from spacer genes

• maturation of crRNA by tracrRNA

• integration of the gRNA into cas9 endonuclease

3. binding viral genome with cas9 complex

4. activity of cas9 complex

5. endogenous DNA repair leading to mutations

what makes CRISPR ideal

• high potency and specificity

• broad application in vivo and ex vivo

• simple editing tools

• ability to adress any site

• ability to target multiple sites simultaneously

• multifunctional

applications of CRISPR

• to understand the role that specific mutations in specific genes influence a particular trait of an organism

• to recreate known stable mutations in cell lines that can serve as models of a particular disease

• to create stable mutations in whole organism and create strains that can be used in research or commerce

• to create gene therapies in order to treat or diagnose congenital diseases, infections, or cancers especially new outbreaks

• to create gene drives that can modify populations of organisms in a specific way

Assessing efficacy

• PCR primers to amplify the region surrounding the target site

• CEL I endonuclease or T7 endonuclease I assay to determine the efficacy of mutagenesis

• DNA sequencing to identify specific mutations

how CRISPR works

1. Cas9 acts as a pair of molecular scissors that cut 2 DNA strands at a specific loaction of the genome

2. A piece of gRNA guides Cas9 to the right part of the genome

3. gRNA has RNA bases that are complementary to those of the target DNA sequence in the genome

4. The cas9 follows the gRNA to the same location in the DNA sequence and makes a cut across both strands of DNA

5. The cell recogonizes the cell is damaged and tries to repair it

on target and off target effects

• CRISPR cas9 can be very efficient (up to 100% mutagenesis)

• off target mutagenesis happens elsewhere in the genome

  • they are most likely to happen at sites with sequence similarity to on target site

CRISPR in dentistry

• new treatment for oral cancer

• inhibition of plaque formation

• reduction/prevention of dental caries and perio disease

• salivary dysfunction