Viral Vectors for gene transfer

What is gene transfer technology?

Gene transfer is defined simply as a technique to efficiently and stably introduce foreign genes into thegenome of target cells. Genes are the basic hereditary units of all life. It is our genes that provide the blueprints necessary to produce all proteins in our bodies, and our proteins that ultimately perform every biological function. Thus, when a gene is stably introduced into a target cell the protein encoded by the gene is produced.

Gene transfer technologies were originally developed as a research tool for investigating gene expression and function. However, as new gene transfer technologies are developed and old technologies refined, the potential 

applications

 have expanded dramatically. Currently, there are a number of gene transfer technologies available which vary greatly in their efficiency of gene transfer and the types of cells they are capable of delivering genes into. Genes can be delivered into cells using lipid-based vectors, naked DNA, electroporation (the application of electrical charge to cells), or viruses which are the most efficient gene transfer vectors.

Recently, gene transfer technology has found its way into clinical applications designed to treat inherited diseases, cancer, and infectious diseases such as AIDS. When genes become altered or damaged so that the encoded protein is no longer functional, as is the case for inherited diseases and many cancers, disease development may occur. Disease development can also be the result of an acquired or infectious disease wherein a pathogenic protein or aberrant protein is produced. In either case, gene transfer technologies can be used to deliver a therapeutic gene into patients. For human clinical applications, gene transfer technologies must be designed that are capable of efficiently delivering genes into primary human cells without harming the recipient. In general, a carrier molecule known as a “vector” is used to deliver the therapeutic gene into target cells. The most commonly used vector for clinical gene transfer applications is a virus which has been genetically engineered to carry the therapeutic gene, but does not have the ability to multiply or cause disease (Figure below).

Lentiviral vectors as gene delivery systems

It is not surprising that the most efficient gene transfer technologies available today exploit the natural ability of viruses to infect and deliver genes into animal cells. Scientists have engineered viruses into gene delivery vectors or carriers that do not cause disease or multiply in infected cells. Oncoretroviruses are the most widely used viral vectors thus far because the retroviral genome inserts into the target cell’s genome following infection. As a result, retroviral vectors can permanently deliver a gene to target cells. However, the use of oncoretroviruses is limited by their ability to infect only dividing cells and by their limited success at establishing stable gene expression in humans. To circumvent these problems, alternative gene transfer technologies are being developed that are capable of delivering genes to a wide range of human cells including non-dividing cells such as neurons. One such gene transfer technology is based on lentiviruses.

Lentiviruses are a family of retroviruses known as slow viruses because symptoms do not generally appear until long after the initial infection. Like all retroviruses, lentiviruses insert their genome into the infected cell’s genome following infection resulting in stable long-term gene expression. Unique to lentiviruses is the ability to naturally infect both dividing and non-dividing cells. Thus, lentiviral-based vectors can be used to deliver genes into non-dividing human cells. This feature greatly expands the scope of potential gene transfer applications.

In developing lentiviral vectors, DNA encoding some or all of the viral genes is removed and replaced with the foreign gene. Thus, the viral vector is designed to be able to enter the cell, deliver the gene, but does not have the ability to replicate or cause disease once inside. The continued development of lentiviral vectors will be critical for the advancement of clinical gene transfer as a viable treatment option for a variety of diseases including genetic disorders and infectious diseases.

GeneCure has developed a lentiviral-based gene transfer technology

GeneCure has developed a patented platform technology of gene transfer based on a primate lentivirus, the simian immunodeficiency virus. This novel gene transfer technology has the ability to efficiently transfer genes into primary human cells without causing harm to humans. This technology will be used to develop vaccines for the prevention and treatment of human diseases.

How GeneCure’s lentiviral-based gene transfer technology works

GeneCure’s patented gene transfer technology is based on a primate lentivirus, the simian immunodeficiency virus (SIV). The company’s lentiviral gene transfer vector (SimVec) is a genetically engineered SIV genome that lacks the genes necessary for viral replication. A therapeutic gene can be introduced into the vector through standard molecular biology techniques. Virus particles encoding the therapeutic gene can be produced in a specialized cell line called a packaging cell line. Virus particles containing the therapeutic gene can then be collected and used to infect and deliver the gene to target cells. Importantly, the virus is capable of infecting and delivering the therapeutic gene to target cells including non-dividing human cells but is unable to multiply and spread to other cells. Because lentiviruses stably integrate into the target cell’s genome, the therapeutic gene can be expressed long-term and is replicated and passed on to all daughter cells during cell division.

Advantages of GeneCure’s gene transfer technology

Efficient gene delivery into primary cells

Current clinical gene transfer is hampered by the lack of effective means to deliver genes into primary human cells. The most commonly used gene transfer technologies in clinical studies are retroviral-based vectors derived from murine retroviruses. Unfortunately, these vectors have limited potential for clinical applications due to their inability to infect non-dividing cells. To address this concern, scientists have taken advantage of lentiviruses, which have the natural ability to infect non-dividing mammalian cells. Lentiviral-based vectors including those based on human immunodeficiency virus, HIV-1, have been developed to deliver genes to non-dividing human cells. However, as HIV-1 is a major human pathogen, the use of HIV-1-based gene transfer technology poses significant concerns for use in human gene transfer applications. GeneCure’s unique technology is based on the simian immunodeficiency virus (SIV), a lentivirus family member that does not cause disease in humans. Thus, GeneCure’s gene transfer technology can effectively deliver genes into human cells including non-dividing and terminally differentiated cells without the risks associated with an HIV-1-based approach.

Safety profile in primate model

In order for viral vectors to be worthy of clinical applications, they must be proven safe in animal models. The primary concern when using retroviruses for human gene transfer applications is the generation ofreplication-competent retrovirus (RCR). Studies performed in rhesus monkeys to monitor formation of replication-competent virus have validated GeneCure’s gene transfer technology as a safe delivery system for future use in clinical studies.

Stable long-term expression of gene

Because lentiviruses permanently integrate into the target cell’s genome, lentiviral vectors allow for stable long-term expression of the gene. Numerous reports demonstrate stable expression of reporter genes for greater than nine months. Additionally, unlike commonly used onco-retroviral vectors, where transcriptional silencing of the gene has been observed in numerous reports, no transcriptional silencing has been observed with lentiviral vectors. Thus, use of lentiviral vectors may overcome the challenges hindering current gene transfer technologies.

No pre-existing immunity

An important consideration when using viral vectors for clinical gene transfer applications is the presence of pre-existing immunity in the target population. Pre-existing immunity occurs when a patient has been previously exposed to the natural virus, as is common for vectors based on the adenovirus (cause of the common cold) and canarypox virus (a harmless relative of smallpox). Previous exposure the virus prepares the patient’s body to quickly mount an immune response should it encounter the virus again. As a result, use of these viral vectors in patients with pre-existing immunity may dampen the effectiveness of the vector due to unwanted immune responses directed at the vector itself. Pre-existing immunity can limit both the ability of the vector to efficiently deliver the gene to target cells as well as limit the duration of gene expression due to immune-mediated destruction of infected cells. In addition, pre-existing immunity can reduce the efficiency of vector re-administration, limiting the usefulness of boosting or re-immunization with the same vector.

GeneCure’s gene transfer technology is based on the simian immunodeficiency virus (SIV), which is not a human pathogen. Accordingly, this technology can be used in clinical gene transfer applications without the problems associated with pre-existing immunity. This includes the ability to re-administer the viral vector to patients as necessary.

Broad host range

GeneCure’s unique gene transfer technology allows production of lentiviral particles which have incorporated the vesicular stomatitis virus G protein (VSV-G) into the viral envelopes. Incorporation of VSV-G envelope protein permits the viral particles to infect a broad range of mammalian and non-mammalian host cells including human, primate, mouse, hamster, and fish cells. This technology greatly expands the applications of gene transfer technology to include studies using cell types which are resistant to infection with other vectors and to non-mammalian studies.

Stable high titer production of viral particles

Many viral-vector mediated gene transfer applications, particularly clinical applications, require a large quantity of viral vector. As a result, viral vectors must be produced at high titers and must be able to withstand further concentration. GeneCure’s unique gene transfer technology allows production of lentiviral particles which have incorporated the vesicular stomatitis virus G protein (VSV-G) into the viral envelopes. Incorporation of VSV-G into viral particles greatly increases the amount and stability of vector produced. Viral vector can be consistently produced at a biological titer of 108 Transducting Units (TU)/ml and can be concentrated to 1011 TU/ml by ultracentrifugation.

Potential applications of GeneCure’s gene transfer technology

GeneCure’s SimVec gene transfer technology has been designed as a multi-use gene product platform. Because the technology can be used to introduce any foreign gene into virtually any cell type, the potential applications are endless.

Clinical gene transfer applications

The most obvious and potentially most beneficial application of GeneCure’s gene transfer technology is the development of gene-based therapeutics to combat human disease. The lentiviral-based gene transfer technology can efficiently and stably deliver virtually any therapeutic gene safely into humanprimary cells. Thus, this technology can be used in clinical gene transfer applications designed to treat any number of human diseases including inherited disease, acquired diseases such as cancer, and infectious diseases. GeneCure will license its lentiviral-based gene transfer technology rights to other organizations developing a wide range of clinical gene transfer products.

Vaccine Development

In addition, GeneCure’s gene transfer technology will allowed for the development of innovative vaccine designs. The technology can be used to generate either preventive or therapeutic vaccines against infectious diseases such as AIDS, SARS, malaria, tuberculosis, hepatitis A, B and C viruses, influenza virus, La Crosse virus, and Ebola virus. GeneCure will be developing its own proprietary gene-based vaccines for the treatment of many human diseases including HIV, Hepatitis B, Hepatitis C, and SARS.

Production of transgenic animals

A transgenic animal is an organism that has had DNA introduced into one or more of its cells artificially. Transgenic animals are produced by introducing the DNA of interest into embryonic stem (ES) cells. ES cells containing the DNA insert are then injected into the embryo of a pregnant female; the embryo is implanted into the female’s uterus and allowed to develop. As the animal develops, every cell derived from the modified ES cells contains the specific DNA insert. In this way, whole animals can be produced with the desired modified DNA. Transgenic animals are powerful tools for studying gene function and testing drugs. This type of technology can be used for the development of transgenic livestock and plants, to produce therapeutic proteins and antibodies on an industrial scale, and even to produce disease-resistant crops.

GeneCure’s lentiviral-based gene transfer technology can be used to efficient deliver the DNA of interest into embryonic stems cells of virtually any cell type including mammalian and non-mammalian cells. Thus, this technology can be used to generate transgenic animals, including those that have proven challenging using other methods.

Gene Silencing using siRNAs

Before a gene can be expressed as its protein product, the DNA encoding the gene is copied into a messenger RNA (mRNA) intermediate. It is this mRNA intermediate that is translated into the final protein product. Interfering with the mRNA intermediate is one method of silencing gene expression and is a powerful tool for genetic analysis in vivo and in vitro. Genes can be silenced through the use of small inhibitory RNAs (siRNAs). siRNAs are small molecules that play a crucial role in the destruction of RNA. siRNAs can be designed to target a specific mRNA and to turn off the expression of a specific gene of interest.

Viral vectors are commonly used to deliver siRNAs into cells or whole animals. GeneCure’s unique lentiviral-based gene transfer technology can be used to deliver siRNAs to mammalian and non-mammlian cells in vitro and in vivo. Even though use of siRNAs is still relatively new, its application has already allowed rapid evaluation of gene functions, and will most likely advance the development of innovative gene-based therapeutics. To date lentiviral-based vectors have been used to deliver stable and targeted siRNAs into primary mammalian cells, stem cells, and transgenic animals.

Stem Cell Manipulation

It has proven particularly challenging to genetically manipulate stem cells. Currently, onco-retroviruses are the most widely used gene transfer vectors. However, onco-retroviruses cannot efficiently deliver genes into stem cells. GeneCure’s lentiviral-based gene transfer technology offers unique biological properties that allow efficient and stable gene delivery into stem cells.

Animal models for human disease

Many human genetic and acquired diseases can be modeled by introducing a gene mutation or an entire gene into a mouse or other animal. While similar genetic manipulations can be achieved in tissue culture cells, expression of the gene in whole organisms provides for a much more comprehensive and physiologically relevant interpretation of the genes normal function. Animal models of human diseases are invaluable tools that provide both a better understanding of the disease and a model in which novel therapeutic approaches can be tested. GeneCure’s lentiviral-based gene transfer technology can be used to produce transgenic animals that model human diseases including diseases that affect non-dividing cells like Parkinson’s disease which affects neurons.

Gene Discovery

Although sequencing of the human genome has been completed, the physiological function of many genes remains to be elucidated. GeneCure’s lentiviral-based gene transfer technology can be used to deliver genes with unknown functions into mammalian cells in order to evaluate their functions. This technology can also be used to screen genes for therapeutic properties, with the aim of discovering novel therapeutic approaches to treat human diseases.

Reporter or marker gene expression in vitro and in vivo

Reporter or marker genes are often introduced into cells or whole organisms in order to track a specific cell population. They are used in countless research applications such as monitoring the spread of tumor cells in animal models of cancer. GeneCure’s lentiviral-based gene transfer technology allows efficient gene delivery into virtually any mammalian and non-mammalian cell type. This technology can be used to deliver a reporter or marker gene into both tissue culture cells and whole organisms.

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Genes Transfer and techniques

Recombination

• Genetic recombination refers to the exchange between two DNA molecules.
  • It results in new combinations of genes on the chromosome.
• You are probably most familiar with the recombination event known as crossing over.
  • In crossing over, two homologous chromosomes (chromosomes that contain the same sequence of genes but can have different alleles) break at corresponding points, switch fragments and rejoin.
  • The result is two recombinant chromosomes.
• In bacteria, crossing over involves a chromosome segment entering the cell and aligning with its homologous segment on the bacterial chromosome.
• The two break at corresponding point, switch fragments and rejoin.
• The result, as before, is two recombinant chromosomes and the bacteria can be called a recombinant cell.
• The recombinant pieces left outside the chromosome will eventually be degraded or lost in cell division.
• But one question still remains...how did the chromosome segment get in to the cell?
• The answer is Genetic Transfer!

Genetic Transfer

• Genetic transfer is the mechanism by which DNA is transferred from a donar to a recipient.
• Once donar DNA is inside the recipient, crossing over can occur.
  • The result is a recombinant cell that has a genome different from either the donar or the recipient.
• In bacteria genetic transfer can happen three ways:
  1. Transformation
  2. Transduction
  3. Conjugation
• Remember that a recombination event must occur after transfer in order that the change in the genome be heritable(passed on to the next generation).

Transformation

• After death or cell lyses, some bacteria release their DNA into the environment.
• Other bacteria, generally of the same species, can come into contact with these fragments, take them up and incorporate them into their DNA by recombination.
  • This method of transfer is the process of transformation.
• Any DNA that is not integrated into he chromosome will be degraded.
• The genetically transformed cell is called a recombinant cell because it has a different genetic makeup than the donar and the recipient.
  • All of the descendants of the recombinant cell will be identical to it.
  • In this way, recombination can give rise to genetic diversity in the population.

Griffith's Experiment

• The transformation process was first demonstrated in 1928 by Frederick Griffith.
• Griffith experimented on Streptococcus pneumoniae, a bacteria that causes pneumonia in mammals.
• When he examined colonies of the bacteria on petri plates, he could tell that there were two different strains.
  • The colonies of one strain appeared smooth.
    • Later analysis revealed that this strain has a polysaccharide capsule and is virulent, that it, it causes pneumonia.
  • The colonies of the other strain appeared rough.
    • This strain has no capsules and is avirulent.
  • When Griffith injected living encapsulated cells into a mouse, the mouse died of pneumonia and the colonies of encapsulated cells were isolated from the blood of the mouse.
  • When living nonencapsulated cells were injected into a mouse, the mouse remained healthy and the colonies of nonencapsulated cells were isolated from the blood of the mouse.
  • Griffith then heat killed the encapsulated cells and injected them into a mouse.
    • The mouse remained healthy and no colonies were isolated.
    • The encapsulated cells lost the ability to cause the disease.
  • However, a combination of heat-killed encapsulated cells and living nonencapsulated cells did cause pneumonia and colonies of living encapsulated cells were isolated from the mouse.
    • How can a combination of these two strains cause pneumonia when either strand alone does not cause the disease?
    • If you guessed the process of transformation you are right!
      • The living nonencapsulated cells came into contact with DNA fragments of the dead capsulated cells.
      • The genes that code for thr capsule entered some of the living cells and a crossing over event occurred.
      • The recombinant cell now has the ability to form a capsule and cause pneumonia.
      • All of the recombinant's offspring have the same ability.
      • That is why the mouse developed pneumonia and died.

Transduction

• Another method of genetic transfer and recombination is transduction.
• This method involves the transfer of DNA from one bacterium to another with the use of a bacteriophage (phage).
  • A phage is a virus that infects bacteria.
  • The phage T4 and the phage lambda, for example, both infect E. coli.
• Because the phage reproductive system is important to understanding transduction, we will briefly review phage lifecycle.
• Phages are obligatory intracellular parasites and must invade a host cell in order to reproduce.
  • T4 multiplies by the lytic cycle which kills the host and lamba multiplies by the lysogenic cycle which does not cause the death of the host cell.
  • In lysogeny, the phage DNA remains latent in the host until it breaks out in a lytic cycle.
• General Steps Of The Lytic Cycle:
  1. Attachment of T4 to receptors on E. coli cell wall.
  2. Penetration of the cell wall by tail core. Inject DNA into host.
  3. E. coli DNA is hydrolyzed. Phage DNA directs biosynthesis of viral parts using the host cell's machinery.
  4. The phages mature as the parts are assembled.
  5. Lyses of E. coli and release of the new phages.
  Watch A T4 Virus Inject Its DNA Into A Bacteria.
• General Steps Of The Lysogenic Cycle:
  1. Phage attaches to E. coli and injects DNA.
  2. Phage circularizes and can enter either the lytic or the lysogenic cycle.
  3. The lytic cycle would occur as previously described.
  4. In the lysogenic cycle the circular phage DNA recombines with E. coli DNA and the phage DNA is now called prophage.
  5. E. coli undergoes cell division, copying prophage and passing to daughter.
     • With more divisions there are more cells with the prophage.
  6. The prophage may exit the chromosome and start a lytic cycle at any time.
• Now that you have reviewed phage lifecycles, we can discuss transduction.
• Transduction can be generalized or specialized.
• The Steps Of General Transduction:
  1. A phage attaches to cell wall of bacterium and injects DNA.
  2. The bacterial chromosome is broken down and biosynthesis of phage DNA and protein occurs.
  3. Sometimes bacterial DNA can be packaged into the virus instead of phage DNA.
     • This phage is defective (can't destroy another host cell) because it does not carry its own genetic material.
  4. The cell lyses, releasing viruses.
  5. The phage carrying bacterial DNA infects another cell.
  6. Crossing over between donor and recipient DNA can occur producing a recombinat cell.
• In generalized transduction, any bacterial genes can be transferred bacause the host's chromosome is broken down into fragments.
  • Whatever piece of bacterial DNA happens to get packaged within the phage is the genetic material that will be transferred between cells.
• In specialized transduction, on the other hand, only certain bacterial genes can be transferred.
  • These genes, as you will see, must exist on either side of the prophage.
  • Specialized transduction requires a phage that uses the lysogenic cycle for reproduction.
  • The Steps In Specialized Transduction:
    1. Remember that in the lysogenic cycle, phage DNA cn exist as a prophage integrated in the bacterial chromosome)
    2. Occasionally when the prophage exits it can take adjacent bacterial genes with it.
    3. The phage DNA directs synthesis of new phages.
       • The phage particles carry phage DNA and bacterial DNA.
    4. The cell lyses, releasing the phages.
    5. A phage carrying bacterial DNA infects another cell.
    6. The joined phage and bacterial DNA circularize.
    7. Along with the prophage, bacterial DNA integrayes with the recipient chromosome by a cross over event.
       • This forms a recombinant cell.

Conjugation

• A third mechanism by which genetic transfer takes place is conjugation.
• This mechanism requires the presence of a special plasmid called the F plasmid.
• Therefore, we will briefly review plamid structure before continuing.
  • Plasmids are small, circular pieces of DNA that are separate and replicate indepentently from the bacterial chromosome.
  • Plasmids contain only a few genes that are usually not needed for growth and reproduction of the cell.
  • However, in stressful situations, plasmids can be crucial for survial.
  • The F plasmid, for example, facilites conjugation.
    • This can give a bacterium new genes that may help it survive in a changing environment.
  • Some plasmids can integrate reversibly into the bacterial chromosome.
    • An integrated plasmid is called an episome.
• Bacteria that have a F plasmid are referred to as as F+ or male.
  • Those that do not have an F plasmid are F- of female.
• The F plasmid consists of 25 genes that mostly code for production of sex pilli.
• A conjugation event occurs when the male cell extends his sex pili and one attaches to the female.
  • This attached pilus is a temporary cytoplasmic bridge through which a replicating F plasmid is transferred from the male to the female.
  • When transfer is complete, the result is two male cells.
• The F plasmid can behave as an episome.
  • When the F+ plasmid is integrated within the bacterial chromosome, the cell is called an Hfr cell (high frequency of recombination cell).
  • The F plasmid always insetrs at the same spot for a bacterial species.
• The Hfr cell still behaves as a F+ cell, transferring F genes to a F-cell, but now it can take some of the bacterial chromosome with it.
• Replication of the Hfr chromosome begins at a fixed point within the F episome and the chromosome is transferred to the female as it replicates.
• Movement of the bacteria usually disrupts conjugation before the entire chromosome, including the tail of the F episome can be transferred.
  • Therefore, the recipient remains F- because the F plasmid is not entirely transferred.
• A cross over event can occur between homologous genes on the Hfr fragment and the F- DNA.
• Pieces of DNA not recombined will be degraded or lost in cell division.
• Now the recombinant genome can be passed on to future generations.Watch DNA Travel Through The Conjugation Tube.

Plasmids

• Plasmids are genetic elements that can also provides a mechanism for genetic change.
• Plasmids, as we discussed previously, are small, circular pieces of DNA that exist and replicate separately from the bacterial chromosome.
• We have already seen the importance of the F plasmid for conjugation, but other plasmids of equal importance can also be found in bacteria.
• One such plasmid is the R plasmid.
• Resistance or R plasmids carry genes that confer resistance to certain antibiotics. A R plasmid usually has two types of genes:
  1. R-determinant: resistance genes that code for enzymes that inactivate certain drugs
  2. RTF (Resistance Transfer Factor): genes for plasmid replication and conjugation.
• Without resistance genes for a particular antibiotic, a bacterium is sensitive to that antibiotic and probably destroyed by it.
• But the presence of resistance genes, on the other hand, allows for their transcription and translation into enzymes that make the drug inactive.
• Resistance is a serious problem. The widespread use of antibiotics in medicine and agriculture has lead to an increasingnumber of resistant strain pathogens.
• These bacteria survive in the presence of the antibiotic and pass the resistance genes on to future generations.
• R plasmids can also be transferred by conjugation from one bacterial cell to another, further increasing numbers in the resistant population.
  

Transposons

• Transposons (Transposable Genetic Elements) are pieces of DNA that can move from one location on the chromosome another, from plasmid to chromosome or vice versa or from one plasmid to another.
• The simplest transposon is an insertion sequence.
  • An insertion sequence contains only one gene that codes frotransposase, the enzyme that catalyzes transposition.
  • The transposase gene is flanked by two DNA sequences called inverted repeats because that two regions are upside-down and backward to each other.
• Transposase binds to these regions and cuts DNA to remove the gene.
• Yhe transposon can enter a number of locations.
  • When it invades a gene it usually inactivates the gene by interrupting the coding sequence and the protein that the gene codes for.
  • Luckil, transposition occurs rarely and is comparable to spontaneous mutation rates in bacteria.
• Complex transposons consist of one or more genes between two insertion sequences.
• The gene, coding for antibiotic resistance, for example, is carried along with the transposon as it inserts elsewhere.
• It could insert in a plasmid and be passed on to other bacteria by conjugation.

Gene Transfer Techniques

The introduction of functional (usually cloned) GENES into cells. A variety of techniques and naturally occurring processes are used for the gene transfer such as cell hybridization, LIPOSOMES or microcell-mediated gene transfer, ELECTROPORATION, chromosome-mediated gene transfer, TRANSFECTION, and GENETIC TRANSDUCTION. Gene transfer may result in genetically transformed cells and individual organisms.

Genetic Therapy

Techniques and strategies which include the use of coding sequences and other conventional or radical means to transform or modify cells for the purpose of treating or reversing disease conditions.

• Genetic Therapies
• Therapies, Genetic
• Therapy, Genetic
• Therapy, DNA
• DNA Therapy
• Genetic Therapy, Somatic
• Genetic Therapies, Somatic
• Somatic Genetic Therapies
• Somatic Genetic Therapy
• Therapies, Somatic Genetic
• Therapy, Somatic Genetic
• Genetic Therapy, Gametic
• Gametic Genetic Therapies
• Gametic Genetic Therapy
• Genetic Therapies, Gametic
• Therapies, Gametic Genetic
• Therapy, Gametic Genetic
• Gene Therapy
• Therapy, Gene
• Gene Therapy, Somatic
• Therapy, Somatic Gene
• Somatic Gene Therapy
• Technique, Gene Transfer
• Techniques, Gene Transfer
• Transfer Technique, Gene
• Transfer Techniques, Gene
• Transgenesis
• Gene Transfer Technique
• Gene Delivery Systems
• Delivery System, Gene
• Delivery Systems, Gene
• Gene Delivery System

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Transformation, Transduction and Transfection –Gene transfer methods

The three very effective modes of gene transfer Transformation, Transduction and Transfection observed in bacteria fascinated the scientist leading to the development of molecular cloning. The basic principle applied in molecular cloning is transfer of desired gene from donor to a selected recipient for various 

applications

 in the field of medicine, research, gene therapy with an ultimate aim of beneficial to the mankind.

Transformation: Transformation is the naturally occurring process of gene transfer which involves absorption of the genetic material by a cell through cell membrane causing the fusion of the foreign DNA with the native DNA resulting in the genetic expression of the received DNA. Transformation is usually a natural method of gene transfer but as a result of technological advancement originated the artificial or induced transformation. Thus there are two types called as natural transformation and artificial or induced transformation. In natural transformation, the foreign DNA attaches itself to the host cell DNA receptor and with the help of the protein DNA translocase it enters the host cell. The presence of nucleases restricts the entry of two strands of the DNA, destroys a single strand thus allowing only one strand to enter the host cell. This single stranded DNA mingles with the host genetic material successfully. 

The artificial or induced method of transformation is done under laboratory condition which is either a chemical mediated gene transfer or done by electroporation. In the chemical mediated gene transfer, the cold conditioned cells in calcium chloride solution are exposed to sudden heat which increases the permeability of the cell membrane allowing the foreign DNA. The electroporation method as the name indicates, pores are made in the cell by exposing it to suitable electric field, allowing the entry of the DNA. The opened up portions of the cell are sealed by the ability of the cell to repair.

Transduction: In transduction, a media like virus is required between two bacterial cells in transferring genes from one cell to the other. Researchers used virus as a tool to introduce foreign DNA from the selected species to the target organism. Transduction mode of gene transfer follows either a lysogenic phase or lytic phase. In the lysogenic phase, the viral (phage) DNA once joining the bacterial DNA through transduction stays dormant in the following generations. The induction of lysogenic cycle by an external factor like UV light results in lytic phase. In lytic phase, the viral or phage DNA exists a s a separate entity in the host cell and the host cell replicates viral DNA mistaking it for its own DNA.As a result many phages are produced within the host cell and when the number exceeds it causes the lysis of the host cell and the phages exits and infects other cells. As this process involves existence of both the genome of the phage and the genome of the bacteria in the same cell, it may result in exchange of some genes between the two DNA. As a result, the newly developed phage leaving the cell may carry a bacterial gene and transfer it to the other cell it infects. Also some of the phage genes may be present in the host cell. There are two types of transduction called as generalized transduction in which any of the bacterial gene is transferred via the bacteriophage to the other bacteria and specialized transduction involves transfer of limited or selected set of genes.

Transfection: One of the methods of gene transfer where the genetic material is deliberately introduced into the animal cell in view of studying various functions of proteins and the gene. This mode of gene transfer involves creation of pores on the cell membrane enabling the cell to receive the foreign genetic material. The significance of creating pores and introducing the DNA into the host mammalian cell contributed to different methods in transfection. Chemical mediated transfection involves use of either calcium phosphate or cationic polymers or liposomes. Electroporation, sonoporation, impalefection, optical transfection, hydro dynamic delivery are some of the non chemical based gene transfer. Particle based transfection uses gene gun technique where a nanoparticle is used to transfer the DNA to host cell or by another method called as magnetofection. Nucleofection and use of heat shock are the other evolved methods for successful transfection.
Gene transfer is a technique to stably and efficiently introduce functional genes (that are usually cloned) into the target cells. Genes are the fundamental hereditary units of all the life forms. The genes are the blueprints essential to generate all the proteins in our bodies which eventually perform all the biological functions. Therefore, when a gene is efficiently introduced into a target cell of the host, the protein which is encoded by that gene is produced.

Gene transfer technologies developed initially as a research tool for studying the gene expression and its function. On the other hand, as novel gene transfer technologies developed and older technologies were sophisticated, its potential applications have expanded significantly. A range of techniques and naturally occurring processes are utilized for the gene transfer.

Chemical Methods:
DEAE-dextran (Diethylaminoethyl-dextran)- It is polycationic compound and is derived from dextran (a polymer of carbohydrate). DEAE-dextran is capable of binding to the anionic phosphodiester backbone of the deoxyribo nucleic acid (DNA) due to its positive charge. The resulting complex retains an overall cationic charge and is capable of binding to the surfaces of negatively charged cell membrane. Consequently, the complex is then taken up by the cell most probably by the process of endocytosis. The advantages of the DEAE-dextran transfection technique include its easiness, reproducibility, and lower cost. 

Calcium Phosphate - Co-precipitation of Calcium phosphate is one of the most famous and broadly utilized techniques for transfection of DNA from the time when it was initially introduced by Van Der Ebin and Graham during early years of 1970’s. This method involves mixing the nucleic acid (DNA) with calcium chloride, and then cautiously adding this mixture to a saline solution with phosphate buffer accompanied by incubating the mixture at room temperature. This produces a precipitate with DNA, which is then spread onto cultured cells. The precipitate is then taken up by the cells by means of phagocytosis or endocytosis. The chief advantages of the calcium phosphate technique are that it is simple, applicable to a wide range of cell types and can be accomplished at lower cost.
Lipofection- It is the most common and generally utilized gene transfer technique in the recent years. Transfection lipids (cationic) are made up of a positively charged head group (for instance amine), a flexible linker group (ether or ester) and 2 or more hydrophobic tail groups. The combined DNA and cationic lipids act instantaneously to form structures called as lipoplexes that are more complex in structure than the simple liposomes. When lipoplexes are prepared under suitable conditions, they sustain an overall positive charge, which enables them to effectively bind to negatively charged cell surfaces and enter the cell by means of endocytosis. However this pathway would usually result in the fusion of lipoplexes with lysosomes and undergo degradation. This problem is overcome by utilizing the neutral helper lipids, for instance dioleoylethanolamine (DOPE), which are generally included along with the cationic lipid. This allows entrapped DNA to escape the endosomes, reach the nucleus and get access to the cell’s transcriptional machinery.

Polymers – In the current era, a diversity of organic polymers are being used to carry out transfection. One of the most well accepted polymer is the, polyethylenimine (PEI). It is a polycationic organic macromolecule that has a high cationic charge density (also called as a proton sponge). It condenses the nucleic acid (DNA) into positively charged particle that interacts with the cell surfaces that are anionic in nature and gains entry into the cells by means of endocytosis. Dendrimers are another group of polymers that are composed of three-dimensional, branched structures known as dendrons. Among them, the polyamidoamine (PAMAM) family of dendrimers have proven to be a useful tool for transfection. Since the sphere-shaped polycationic dendrimers are alike in proportion and shape to the histone clusters, they can compact the DNA to a small size and facilitate its entry into cells.

Targeting Proteins & Peptides - A range of protein and peptide sequences have been utilized to target, enhance or mediate delivery of nucleic acids in a large variety of applications. Such proteins and peptides are regularly utilized along with cationic lipids (example integrin-targeting peptide, Fusogenic peptides such as GALA, N-terminal peptide of influenza hemagglutinin HA2 subunit). The key benefits of utilizing targeting proteins and peptides are that both will enhance the transfection efficiencies and provide targeted delivery.

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Bacterial Gene Transfer

Bacterial Gene Transfer

Bacterial gene exchange differs from eukaryotes:

• Bacteria do not exchange genes by meiosis.  (Why not?)  They rarely exchange two entire genomes.
• Bacteria commonly exchange small pieces of genome, a few genes at a time, through transformation, transduction, or conjugation.
• Transfer between species, even kingdoms, is common; less common  in eukaryotes, though it does occur.

Transformation.
Transformation is the uptake of DNA from outside the cell.  Only a single strand is taken up, through a special protein  complex in the cell membrane.  The process requires calcium ion (Ca2+).  Transformation occurs at extremely low frequency, but with large populations of bacteria, it offers a significant route for genetic transfer.

Phage Transduction
There are two types:

• Generalized transduction (depicted below).  A piece of host DNA gets packaged by mistake, instead of the phage DNA.  This rare event results in a phage delivering only bacterial DNA to the next host.  The DNA then recombines homologously, replacing the host allele.
• Specialized transduction, in which a lysogenic prophage recombines itself out of the genome (bysite-specific recombination) and mistakenly includes a piece of bacterial DNA.  The resulting phage progeny can infect cells to produce lysogens with a second copy of the allele they had packaged, attached to the phage DNA.

Diagram of generalized transduction:

Plasmids

Plasmids are small circles of DNA that contain an origin of replication (ori) and a small number of genes, some of which may confer a survival advantage on a host.  Some plasmids can transfer between different species; even between different kingdoms.  A shuttle vector is a plasmid engineered in the test tube to contain an ori site for bacteria, and an ori site for animal or plant cells.  Shuttle vectors are enormously useful to clone a gene conveniently in bacteria, then express it in tissue culture.

Conjugation

Conjugation is the process by which a plasmid is transferred from an F+ cell into an F- cell.  The F factor in the F+ cell contains genes which express pili for attachment, and special membrane proteins for the transfer complex.  Some conjugative plasmids carry drug resistant strains--a big problem for hospitals.

If an F plasmid is integrated into a host genome (an Hfr, for high frequency recombination) the F factor can transfer part or all of the genome into the recipient F- cell.

I 
Electronic Companion to Genetics, Cogito Learning MediaI Episomes and Hfr 
The F plasmid can recombine itself into the host chromosome by site-specific recombination.  It can then (a) transfer part or all of the chromosome into a recipient F- cell, as an Hfr; or (b) recombine itself out again, and mistakenly pick up a piece of the host chromosome to carry into the next F- host.

Problem (5)  Explain two different genetic processes in bacteria that can create a "partial diploid" for a small part of the genome.  Explain why these processes are useful for bacterial genetic analysis.

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Mobile genes.
Some genes, such those encoding resistance to antibiotics, can move from one genome to another, at anew place in the genetic map.  Some of these mobile genes can even transfer between two distantly related species of organism.

Transposable elements
The first transposable elements to be characterized genetically were controling elements for seed coat color in maize (corn.)  Barbara McClintock won the Nobel Prize for showing that DNA is not all "fixed" in the genome, but that some sequences can insert and excise by intramolecular recombination.

There are  many classes of transposons.  In bacteria, a common structure of a transposon contains:

• An insertion sequence (IS) at the right and left ends.  The IS contains the gene encoding the transposase enzyme.
• A gene encoding antibiotic resistance.  This gene confers a selective advantage to bacteria containing the transposon, in  the presence of the antibiotic.

Transposons

Some bacterial transposons can be exchanged among many different species, usually carried byplasmids.
Other transposed pieces of DNA can be inverted at one place in one species, to turn on or off the regulation of a gene.  An example of such a site-specific transposition event is the flagellar gene regulation catalyzed by hin recombinase.

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Genomic Islands

Bacterial genomes often contain "islands" of DNA transferred relatively recently from another species. The "genomic island" may confer special properties to a pathogen, or two a strain inhabiting a special niche in an ecosystem.

Herbert Schmidt and Michael Hensel, Clinical Microbiology Reviews, January 2004, Vol. 17, p. 14-56.

A pathogenicity island is a genomic island that converts a "harmless" bacterium to a pathogen. The pathogenicity island is a distinct region of DNA present in the genome of pathogenic bacteria but absent in nonpathogenic strains of the same species. Transfer may be mediated by an integrase enzyme (int). The island is typically inserted at a tRNA gene in the core genome. The pathogenicity island typically contains virulence genes (V1 to V4) interspersed with other mobility elements, such as insertion sequences (IS). How do we recognize a recently inserted pathogenicity island? One clue typically is a difference in percent GC content, compared to the core genome.

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Examples of Pathogenicity Islands

A. The cag island of H. pylori (cause of stomach ulcers) harbors genes for a type IV secretion system that can translocate the toxin CagA into human cells, causing an inflammatory response. B. The SP-1 island of Salmonella typhimurium (typhus and food poisoning) encodes a type III secretion system (grey), secreted proteins (dark grey), and regulatory proteins. The same island includes metabolic proteins unrelated to virulence. C. The HPI island of Yersinia enterocolitica has genes that encode a high-affinity iron uptake system (dark grey) needed for extracellular growth of the pathogen during colonization of the host. D. The vSAL island of multiple drug-resistant Staphylococcus aureus (MRSA) encodes a remarkably high number of enterotoxins.

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Genomic islands in two strains of a marine phototroph, Prochlorococcus

The two strains of Prochlorococcus marinus are cyanobacteria, major oxygenic 

producers

 in the oceans, consuming a large part of atmospheric CO₂. The strains MED4 and MIT96512 differ by only 0.8% of their genome, yet their distributions throughout the ocean are very different, for unknown reasons. The reason for the difference in distribution may have to do with genes encoded within five genomic islands specific to MED4 (ISL1, ISL4, ISL5) or to MIT9312 (ISL2, ISL3).


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