Human somatic gene therapy - concepts and applications

Human gene therapy can be defined as the transfer of exogenous genes or nucleotide sequences into somatic cells for the purpose of preventing, correcting or healing various diseases.

Somatic gene therapy is a new type of weapon in the fight against acquired diseases.DNA is considered as a drug providing a framework for curing thousands of genetic diseases. It also satisfies one of the greatest dreams of clinical medicine: ‘molecular surgery’ at the root of the disease.

Cystic fibrosis (CF)

People suffering from cystic fibrosis lack CF gene needed to produce a salt-regulating channel protein, cystic fibrosis transmembrane conductance regulator (CFTR). This protein regulates the flow of chloride in to epithelial cells that cover the air passages of the nose and lungs. Without this regulation, patients with CF disease build up thick mucus that makes them prone to chronic lung disease. The gene therapy technique to correct this abnormality might employ an adenovirus to transfer a normal copy of the CFTR gene. The gene is introduced into the patient by spraying into the nose.

Familial hypercholesterolemia (FH)

The patients with genetic disorder unable to process cholesterol properly by a low density lipoprotein receptor(LDLR), which leads to high levels of fat in the blood stream.  Patients with FH disease suffer from heart attacks and strokes because of blocked arteries. A gene therapy approach is developed with partial removal of patient’s liver. Corrected copy of a gene is inserted into liver sections, which are transplanted back into patients. The healthy gene may reduce the cholesterol build-up in the blood of the patient.

Duchene muscular dystrophy (DMD)

DMD is the most common childhood form of muscular dystrophy because of the failure to express dystrophin in the muscle fibres. It is a lethal, recessive X-linked disorder, more frequent in males. Muscular dystrophy is characterized by progressive muscle weakness, defects in muscle proteins and death of muscle cells and tissue. Viral mediated in vivo gene transfer method was developed to deliver dystrophin gene to patient’s muscle. Simple non-viral gene transfer in vivo was also developed using naked plasmid DNA.

Cancer therapy

In general, cancers have at least one mutation to a proto-oncogene (yielding) an oncogene) and at least one to a tumour suppressor gene allowing the cancer to proliferate. Oncogene inactivation may be targeted at the level of DNA, RNA transcription or protein product. Oligonucleotides are designed in sequence specific manner to target the promoter regions of oncogenes. At the RNA level, antisense techniques prevent transport and translation of the oncogene mRNA by providing a complementary RNA molecule (e.g., C-myc gene). Ribozymes, antisense oligonucleotides with a cleavage action will reduce the stability of oncogene mRNA. Restoration of the tumour suppressor gene such as p53 can be sufficient to cause cellular apoptosis and arrest tumour growth.

Molecular chemotherapy for cancer cells

Herpes simplex virus thymidine kinase (HSV-Tk) converts the prodrug ganciclovir into toxic metabolites. This toxicity is sufficient enough to kill the cancer cells.

DNA repair by gene therapy

Another approach involves the repairing of a gene using chimeric oligonucleotides. Homologous recombination is a natural process that controls the replacement of a defective gene. Highly precise DNA repair is performed by using DNA oligonucleotides to introduce site specific changes in the genome, even a single incorrect base can be corrected.

Transplantation tolerance in organ transplanted patients

In organ transplanted patients, the use of immunosuppressive drug is associated with increased risk of the development of cancer, infectious and ischemic heart disease. Gene therapy can be used to reduce the immunogenicity by introduction of genes to block T-cell activation.

HIV treatment by gene therapy

By introducing an antiviral gene into an infected T-lymphocyte, the HIV virus can be killed or expressing an antiviral gene in the normal T-lymphocyte may protect the patient from future HIV infection.

Replacement of hormones and blood factors by gene therapy

If an erythropoietin gene or factor IX gene is transferred via adeno-associated vector into muscles, it will cause the expression of relevant protein.

Prodrug activation

In herpes simplex virus thymidine kinase method, the thymidine kinase enzyme in every transduced cell converts the prodrug ganciclovir into monophosphate and triphosphates forms. The triphosphate form of ganciclovir interferes with the DNA replication and kill  the cancer cells.

Treatment of hepatitis B and C

The genetic sequence of RNA molecules of the particular virus is cut and destroyed by using ribozyme producing genes.

Nucleic acid probes - characteristics, probe-assays and applications

Nucleic acid probes are used to detect specific sequence of target DNA or RNA molecules from a mixture.  The single stranded soluble nucleic acid probe hybridizes to the complementary target DNA or RNA that is immobilized on a nitrocellulose or nylon membrane. The nucleic acid hybridization is used for a variety of biotechnological applications such as the detection of cloned DNA, analysis of genetic organization and the diagnosis of genetic diseases. Nucleic acid hybridization is rapid, specific and reliable between complementary strands.

         Definition

In DNA technology, a labelled single stranded nucleic acid molecule is used to tag a specific complementary nucleotide sequence in a nucleic acid sample.

         Kinds of nucleic acid probes

1.    Genomic DNA probes – using probes, more copies of fragments of genomes are obtained, purified and labelled.

2.    cDNA probes – obtained from mRNA using RTase enzyme and labelled.

3.    Synthetic oligonucleotide probes – made up of 14-20 base pairs long.

4.    RNA probes –to locate RNA molecules and was synthesized in vitro using phage RNA polymerase.

Characteristics of DNA probes

1.    DNA probes are very specific in their hybridization wit particular complementary DNA sequence.

2.    DNA probes never produce false positive or false negative results.

3.    DNA probes are more stable and resistant to heat and chemicals. They can be used to characterize DNA from dead tissues.

4.     The size of the DNA probes range from 13 – 100bp.

5.    With the help of PCR, DNA sequences can be amplified up to million folds.

              Labels of DNA probes

·       Labelled with radionuclides-P³², I¹²⁵, S³⁵, H³

·       With fluorescent labels – fluorescein, rhodamines, ethidium, rare earth chelates.

·       With luminescent labels – luminal derivatives, acridium esters, luciferase.

·       With enzyme markers – alkaline phosphatase, horse radish peroxidise.

·       With the combination of above labels

Preparation of DNA probes

The DNA sequence of interest is first cloned into a suitable plasmid. Then the plasmid with the cloned gene is transformed into appropriate host cells to amplify the plasmid. The transformant host cells are then selected using biomarker like antibiotic resistance of plasmids. The transformant host cells are separately cultured to get more copies of plasmid – DNA construct. Then the host bacterial cells are harvested and lysed in order to isolate the plasmid DNA with the cloned gene. The plasmid DNA lysate is precipitated differentially and centrifuged in Cesium chloride density gradients using ethidium bromide to purify plasmid DNA-DNA construct. Plasmid is purified and used for preparing DNA probe by incorporating appropriate label. The cloned gene can be retrieved by REase. The enzyme DNAase is used to cause nicks and DNA polymerase I is used to incorporate radio-labelled (P32 – dCTP) deoxyribonucleotides.  The DNA is denatured using sodium chloride to free out unincorporated deoxyribonucleotides from DNA’ by column chromatography using sephadex 600. Two peaks one with labelled DNA and other for P32 –dCTP are obtained. The first peak is separately collected.

DNA probe assays

The assay consists of 4 steps such as sample preparation, hybridization, separation and detection.

Sample preparation - the samples like blood, CSF, urine, stool or tissues are collected from pathogenic organisms. The separation of the target DNA molecules requires elaborate, time consuming multistep mechanical and chemical extraction procedures. The target molecules may be increased by PCR or LCR etc.

Hybridization – Specific DNA probes are added to the DNA derived from the sample and allowing complementary DNA to join. It may be carried out either as heterogeneous or homogeneous process.

Heterogeneous process uses solid support to immobilize target DNA. Examples are dot blot or in situ hybridization. Homogeneous process occurs in solution and after hybridization event, hybrid DNA is usually immobilized onto a support like filter or beads. Homogeneous process allows rapid and more efficient hybridization.

Separation – column chromatography, electrophoresis or paramagnetic beads may be used to recover true hybrids.

Detection techniques – radioisotopic, flurorescent or chemical luminescent techniques may be used. The probes are appropriately labelled with radioisotopes (I125, P32, S35 or H3) or flurorescent dyes or enzymes.

Applications  of DNA probes

Genetic engineering research - DNA probes are used to identify the clones possessing specific DNA sequences and to identify transformants possessing specific genetic characters. The probes can be used in basic studies in molecular biology, population genetics, and ecological genetics.

Disease diagnosis/epidemiological studies – DNA probes are used to identify genetic or microbial diseases or changes in the DNA of brain tumours.

Identification of food contaminants – it is used to identify pathogenic viruses in imported food samples.

Forensic investigations – Probes are used to identify criminals, solving disputed paternity cases, establishing family relations and solving immigration cases.

Agricultural applications – Probes are used to identify good varieties of seeds or plants.

Taxonomy - Probes are used to identify organisms at species or strain level.

DNA probes are used in anthropological survey of human races, and ante-natal diagnosis of inherited diseases.

Recombinant DNA technology - methods and applications

Recombinant DNA technology is modifying the genetic makeup of an organism either by adding new genes or by changing the existing genes. It is a technique of preparing r DNA in vitro by cutting up DNA molecules and splicing the fragments together from more than one organism. Recombinant DNA is a form of artificial DNA that is made through the combination or insertion of one or more DNA strands.  Recombinant DNA is also referred to as "chimera."

Using recombinant techniques, particular sequences of DNA can be isolated, manipulated, and re-introduced into many different kinds of living things. The desired results are improvement of microorganisms, plants, and animals, for a particular purpose.

Objectives of  r DNA Technology

• Artificially synthesize new genes. 
• Altering the genome of an organism.
• Bring about new gene combinations not found in nature. 
• Understanding the hereditary diseases and their cure.
• Improving human genome.

Enzymes are the chemical knives in r DNA technology

DNA or RNA polymerase-replicating or annealing a DNA chain.
Reverse transcriptase – synthesize c DNA from RNA template.
DNA ligase – joining DNA strands together.
Nuclease-breaks phospho-diester bonds within free ends (exonucleases) or in an interior position (Endonucleases ).
Restriction endonuclease – recognizes a specific base sequence and cuts the DNA.

Restriction endonucleases (RE ases)

Restriction endonuclease is a special class of sequence –specific enzymes.It is found in bacteria which protect its genetic material from the invasive attacks of viruses. It is a site-specific enzyme which cleaves DNA molecules only at specific nucleotide sequences. REases recognize DNA base sequences that are palindromes. REases make two single stranded breaks, one in each strand. REases make staggered cuts with complementary base sequences for easy circularization.

Types of Vectors

o   Bacterial plasmid vectors

o   Bacteriophage vectors

o   Cosmid vectors

o   Expression vectors

o   Bacterial Artificial Chromosomes (BAC)

o   Yeast Artificial Chromosomes (YAC)

o   Ti  and Ri vectors

Plasmids

Plasmids are relatively small, self-replicating duplex extra-chromosomal DNAs maintained as independent molecules. Lederberg coined the term ‘plasmid’  in 1952. They are found in bacteria, yeast and streptomyces. Plasmids range in size from < 1 Kb to 500Kb.

Phage Vectors

Two types of phage vectors have been extensively developed-λ and M13. Phage vectors have engineered phage genomes previously genetically modified to include restriction sites. After insertion of foreign DNA, the recombinant phage genome is packaged into the capsid and used to infect host cells

Cosmids

Hybrid vector constructed to contain features from both phages and plasmids. Cosmids  have a selectable marker, multiple cloning sites from plasmids and a cos site from l phage

Artificial Chromosomes

Large fragments of DNA  can  be cloned in artificial chromosomes. Mapping of genes is easier. Artificial chromosomes have played important role in the human genome project. One copy of YAC is present per cell. E.g. yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs)

Host cell types

• Prokaryotic hosts – Bacteria, E . Coli, Bacillus sp., Pseudomonas sp., Streptomyces sp.
• Eukaryotic hosts - Yeast – Saccharomyces, Fungi- Aspergillus, Neurospora, Algae - Chlamydomonas

Two types of host-vectors

• Cloning vector - Propagation of DNA inserts
• Expression vector - Production of proteins

Molecular Cloning / DNA cloning

Molecular cloning refers to the process of making multiple DNA molecules.

Step  1– fragmentation -breaking apart a strand of DNA

Step 2 – ligation-gluing together pieces of DNA in a desired sequence.

Step 3 –Transfection -  inserting the newly formed DNA into cells.

Step 4-Screening / selection – selecting out the cells that were successfully tranfected with the new DNA

Gene transfer technology

• Transduction- Virus mediated gene transfer.
• Tranfection - Chemical or physical tricks to persuade cells to take DNA from the culture medium.
• Direct transfer - Physically inserting the gene e.g. microinjection
• Natural gene transfer - A receptor – mediated lateral binding fusogenic proteins used.

Calcium phosphate –co precipitate  method

This method was described by Graham and Van der Eb in 1973.

It is a process for inserting foreign DNA into bacteria. The bacterial cells are treated with  ice-cold calcium chloride. The plasmid DNA is added  to cells chilled on ice which form calcium phosphate –DNA precipitate. The cell and DNA mixture is heated  to 42^oC. The membrane becomes fluid and plasmid DNA enters bacterial cells and is replicated and expressed

Electroporation

It  involves a brief application of high voltage electric current to the cells resulting in the formation of transient holes in the cell membrane through which plasmid DNA can enter the cell. The transformation efficiency is high. Quick restoration of membrane fluidity and closing of pores is crucial for survival of cell after the pulse.

Selection  techniques for rDNA molecules

•         DNA hybridization assay

•         Colony immunoassay

•         Screening by protein activity

•         Genetic screening methods

Process of selection

Selection is a process designed to facilitate the identification of recombinant bacteria while preventing the growth of non-transformed bacteria. After transformation, the bacteria are challenged with an antibiotic (such as ampicillin). If the E. coli have taken up and expressed an ampicillin resistance gene on a plasmid, they will live - otherwise they will die. This process is called selection because selected bacteria may survive.

DNA hybridization assay

This technique was introduced by Grunstein and Hogness (1978). The target DNA is denatured at 800C and bound to a nitrocellulose filter discs. Such filters are hybridized with radioactive DNA probes. The results are monitored by autoradiography.

Colony immunoassay

The transformed colonies are transferred to a nitrocellulose filter. The colonies are lysed and the released proteins are attached to the matrix. The matrix is treated with a primary antibody which specifically binds to the proteins encoded by the target gene. Then the matrix was washed to remove any unbound antibody. Then the matrix was treated with a second antibody which was an enzyme, alkaline phosphatase. The target protein (antigen) was treated with a colorless substrate. The colorless substrate is hydrolyzed by the alkaline phosphatase into a colored complex.

The Tools of Recombinant DNA Technology

—  Gene Libraries

The collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host. The library may contain all genes of a single chromosome. Screening, identification and characterization of cloned fragments are possible with suitable probes.

cDNA Libraries

The library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. mRNA from tissue of interest is isolated and converted into a double-stranded DNA by using the enzyme reverse transcriptase. The newly synthesized molecules are called complementary DNA (cDNA) because it is an exact copy of the mRNA.

Supporting techniques of Recombinant DNA Technology

      Multiplying DNA in vitro by Polymerase Chain Reaction (PCR)

It was developed in the 1980s by Kary Mullis. This technique is used for making copies, or amplifying, a specific sequence of DNA in a short period of time. It is a repetitive process consisting of three steps: Denaturation, Priming and Extension.It can be automated using a thermocycler. At the end of one cycle, the amount of DNA has doubled. Cycles are repeated 20–30 times

      Separating DNA Molecules by Gel Electrophoresis

The molecules are separated on the basis of electrical charge, size, and shape. It allows to isolate DNA of interest. Negatively charged DNA drawn toward positive electrode. Agarose makes up gel; acts as molecular sieve. Smaller fragments migrate faster than larger ones.Size is determined by comparing distance migrated to standards.

Applications of rDNA technology

Production of transgenic organisms - Recombinant plants and animals altered by addition of genes from other organisms. Transgenic plants that can produce their own insecticides e.g.Better Crops (drought , heat and salt resistance)
Recombinant DNA technology has been used for creating new animal species using the cloning technology. rDNA technology is used to elucidate molecular events in biological processes like cell differentiation, aging etc.
Recombinant Vaccines (ie. Hepatitis B
Gene therapy - Missing or defective genes replaced with normal copies e.g. sickle cell anaemia and severe combined immuno-deficiency (SCID).
Large-scale production of human proteins by genetically engineered bacteria such as : insulin, Growth hormone, Interferons and blood clotting factors (VIII & IX)

Disadvantages of Recombinant technology:

Recombinant technology  can be commercialized and became big source of income for businessmen. The products have effects on the natural immune system of the body. The transgenic organisms can destroy natural ecosystem that relies on organic cycle. They are prone to undergo mutation that could have harmful effects. There is possibility of manufacturing of biological weapons such as botulism & anthrax to target humans with specific genotype. There is a concern of creating super‐human race.

Polymerase Chain Reaction (PCR) - methods and applications

Polymerase chain reaction is a biochemical technique used in Experimental Biotechnology to amplify a specific fragment of target DNA. PCR is a novel molecular procedure based on thermal cycling which consist of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of defined DNA sequences.
PCR was discovered by Kary B. Mulis in 1983 of Cetus Corporation, a Biotech company in California, USA. He won the Nobel Prize for Chemistry in 1993 for ‘contributions to the developments of methods within DNA-based chemistry’. ‘Taq polymerase’ an enzyme used in PCR was described as ‘molecule of the year’ 1989. PCR is now an indispensable technique used in medical and biological research labs for a variety of applications.

A copying machine for DNA molecules

PCR multiplies a single, microscopic strand of the DNA molecule into billions of times within hours. PCR has a major impact on recombinant DNA technology. PCR has multiple applications in medicine, genetics, biotechnology, and forensics.
PCR-A DNA multiplication protocol
PCR is a powerful technique, in which from a single copy of a DNA molecule, millions of copies can be obtained with high accuracy, specificity and in a very short time. DNA amplification process in PCR is cyclical and the concentration of DNA doubles at each cycle. The total amount of DNA concentration increases exponentially during the cyclical process of PCR machine.

The ‘master mix’ components for PCR machine

· A thermostable DNA polymerase: tag polymerase
· A template DNA
· A complete set of deoxynucleotide triphosphates e.g. dATP, dCTP, dGTP and dTTP
· Tris buffer of pH 8.8
· A pair of oligonucleotide primers
· Mg 2+ and detergents
· 2-mercaptoethanol to stabilize proteins during thermal cycle.

Requirements for PCR

·        DNA template – DNA segment to be amplified.

·    Two primers- a short segment of DNA (forward and reverse primers) about 20–25 bases long.

·    Taq polymerase – an enzyme to synthesize DNA copies.

·     Deoxynucleotide triphosphates – the building blocks for new DNA strand.

·     Buffer solution – a suitable chemical environment.

·     Divalent cations – Mg ²⁺ ions

·     Monovalent  ions – Potassium ions

·     PCR machine – a thermal  cycler

Thermostable DNA polymerase

The thermophilic DNA polymerases catalyze template-directed synthesis of DNA from nucleotide triphosphates.

Several thermostable polymerase enzymes are used in PCR

•         Pfu DNA polymerase obtained from - Pyrococcus furiosus

•         Vent polymerase obtained from- Thermococcus litoralis

•         Taq polymerase  obtained from - Thermus aquaticus

Oligonucleotide primers

They are synthesized chemically to be complementary to sequences which flank the region of DNA to be amplified. They are usually about 20-25 nucleotides in length. The primers are designed to anneal specifically to the opposite strands of the template molecule. It is the specificity of the primer annealing reaction which ensures that the PCR amplifies the appropriate region of the template DNA.

Critical steps in PCR

1.     Sample Preparation

2.     Target selection

3.      Primer selection

3 – Temperature cycle in PCR

•         The temperature - 90-98⁰C separates two strands of target DNA.

•         The temperature– 40-60⁰C anneals two complementary primers to the ends of separated single strands of target DNA. 

•         The temperature 72⁰ C allows taq polymerase to use ss target DNA and primers to synthesize new strands.

PCR protocol

1. Denaturation of ds DNA template –melting of target DNA-it is the thermal denaturation of the dsDNA molecules at 950C for 1 min.
2. Annealing of two oligonucleotide primers at 680C for 60 sec. The annealing temperature is dependent on the length and G+C content of the primer sequences.
3. Extension of dsDNA molecules – temperature is raised to 750C for about 30 sec.
The step cycle programme makes the instrument to heat and cool to the set temperatures due to solid state Peltier-effect device, which actively modulates the desired temperature. There may be as many as 30-35 cycles.

Variants of PCR

·    In standard PCR, the sequences of both ends of target DNA have to be known. Two primers define the ends of target DNA and only that part is amplified.

·   In single sided PCR, the DNA is rearranged before amplification so that only one primer is needed. This is also called Anchored PCR.

·   In inverse PCR, the DNA at primer sites rather than between two primers is amplified because primer sites which are bracketing may have important sequence like promoter for triggering target gene into action.

Characterization of PCR product

•        Contamination of the reagents by foreign DNA or annealing of primers to alternative sites in the template DNA may produce unwanted DNA molecules.

•        Multiple bands in the electropherogram suggest primers annealing to multiple sites.

•        Smear of DNA suggests presence of excess template DNA.

Problems and limitations

• Contamination of reaction mixture by bacteria, viruses, and our own DNA presents a real problem.
• PCR cannot substitute for cell- based gene cloning, when large amounts of a gene are desired.
• Taq polymerase used in PCR often lack 3' to 5' exonuclease activity. This enzyme lacks the ability to correct mis-incorporated nucleotides.
• PCRs of longer products are less efficient due to enzyme activity loss. PCR can be applied only to short DNA fragments.

Applications of PCR

• Detection of pathogens in food, water and tissue specimens.
• Detection of tuberculosis, AIDS and other microbial diseases.
• Diagnosis of genetic diseases-e.g. sickle cell anemia, β-thalasemia, hemophilia
• Identification of criminals, disputed parentage
• Monitor gene expression in genetic engineering or gene therapy experiments.
• To study genetic profile of animals and to trace evolutionary and cultural lineage of human beings.
• To study DNA polymorphism.
• To determine orientation and location of restriction fragments relative to one another.
• To conduct microbial surveillance of the environment.

References

•        Saiki, R., Scharf, S., Faloona, F., Mullis, K., Horn, G., and Erlich, H. (1985). Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 230: 1350-54

•        Mullis, K. and Faloona, F. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155: 335-350.

•        Mullis, K. (1990). The unusual origin of the polymerase chain reaction. Scientific American April 56-65

•        Rabinow, P. (1996). Making PCR: A story of biotechnology. University of Chicago Press.