DNA vaccination

From Wikipedia, the free encyclopedia

The making of a DNA vaccine.

DNA vaccination is a technique for protecting an organism against disease by injecting it with genetically engineered DNA to produce an immunological response. Nucleic acid vaccines are still experimental, and have been applied to a number of viral, bacterial and parasitic models of disease, as well as to several tumour models. DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a wider range of immune response types. Vaccines are among the greatest achievements of modern medicine in industrial nations, they have eliminated naturally-occurring cases of smallpox, and nearly eliminated polio, while other diseases, such as typhus, rotavirus, hepatitis A and B and others are well controlled.[1] Conventional vaccines, however, only cover a small number of diseases, and infections that lack effective vaccines kill millions of people every year, with AIDS, hepatitis C andmalaria being particularly common. First generation vaccines are whole-organism vaccines either live and weakened, or killed forms.[2] Live, attenuated vaccines, such as smallpox and polio vaccines, are able to induce killer T-cell (TC or CTL) responses, helper T-cell (TH) responses and antibody immunity. However, there is a small risk that attenuated forms of a

pathogen can revert to a dangerous form, and may still be able to cause disease in immunocompromised people (such as those with AIDS). While killed vaccines do not have this risk, they cannot generate specific killer T cell responses, and may not work at all for some diseases.[2] In order to minimise these risks, so-called second generation vaccines were developed. These are subunit vaccines, consisting of definedprotein antigens (such as tetanus or diphtheria toxoid) or recombinant protein components (such as the hepatitis B surface antigen). These, too, are able to generate TH and antibody responses, but not killer T cell responses. DNA vaccines are third generation vaccines, and are made up of a small, circular piece of bacterial DNA (called a plasmid) that has been genetically engineered to produce one or two specific proteins (antigens) from a pathogen. The vaccine DNA is injected into the cells of the body, where the "inner machinery" of the host cells "reads" the DNA and converts it into pathogenic proteins. Because these proteins are recognised as foreign, when they are processed by the host cells and displayed on their surface, the immune system is alerted, which then triggers a range of immune responses.[1][2] These DNA vaccines developed from failed gene therapy experiments. The first demonstration of a plasmid-induced immune response was when miceinoculated with a plasmid expressing human growth hormone elicited antibodies instead of altering growth.[3]
Contents
[hide]

1 Current use 2 Advantages and disadvantages of DNA vaccines 3 Plasmid vectors for use in vaccination

o o

3.1 Vector design 3.2 Vaccine insert design

4 Delivery methods 5 Immune response raised by DNA vaccines

5.1 Helper T-Cell responses

o o

5.1.1 Raising of different types of T-cell help 5.1.2 Mechanistic basis for different types of T-Cell help 5.1.3 Practical uses of polarised T-Cell help

5.2 Cytotoxic T-cell responses 5.3 Humoral (antibody) response

5.3.1 Kinetics of antibody response

6 Mechanistic basis for DNA raised immune responses

o o o o o

6.1 DNA Uptake Mechanism 6.2 Antigen presentation by bone marrow-derived cells 6.3 Role of the target site 6.4 Maintenance of immune response 6.5 Interferons

7 Modulation of the immune response

o o o o

7.1 Cytokine modulation 7.2 Immunostimulatory CpG motifs 7.3 Alternative boosts 7.4 Additional methods of enhancing DNA-Raised immune responses

8 See also 9 References

7.4.1 Formulations of DNA 7.4.2 Alphavirus vectors

10 External links

[edit]Current

use

Thus far, few experimental trials have evoked a response sufficiently strong enough to protect against disease, and the usefulness of the technique, while tantalizing, remains to be conclusively proven in human trials. However, in June 2006 positive results were announced for a bird flu DNA vaccine [4] and a veterinary DNA vaccine to protect horses from West Nile virus has been approved.[5][6] In August 2007, a preliminary study in DNA vaccination against multiple sclerosis was reported as being effective.[7]
[edit]Advantages

and disadvantages of DNA vaccines

Table 1. Advantages And Disadvantages Of Nucleic Acid-Based Immunization

Advantages Subunit vaccination with no risk for infection[1] Antigen presentation by both MHC class I and class II molecules
[1]

Disadvantages

Limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides)

Able to polarise T-cell help toward type 1 or type 2[1] Immune response focused only on antigen of interest Ease of development and production
[1]

Risk of affecting genes controlling cell growth

Possibility of inducing antibody production

Stability of vaccine for storage and shipping Cost-effectiveness Obviates need for peptide synthesis, expression and purification of recombinant proteins and the use of toxic adjuvants[8]

against DNA Possibility of tolerance to the antigen (protein) produced Potential for atypical processing of bacterial and parasite proteins[1]

Long-term persistence of immunogen[2] In vivo expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications[2]

[edit]Plasmid [edit]Vector

vectors for use in vaccination

design

DNA vaccines elicit the best immune response when highly active expression vectors are used. These are plasmids which usually consist of a strong viral promoter to drive the in vivo transcription andtranslation of the gene (or complementary DNA) of interest.[9] Intron A may sometimes be included to improve mRNA stability and hence increase protein expression.[10] Plasmids also include a strongpolyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences.[1][2][11] Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.[12] Because the plasmid is the vehicle from which the immunogen is expressed, optimising vector design for maximal protein expression is essential.[12] One way of enhancing protein expression is by optimising the codon usage of pathogenic mRNAs for eukaryotic cells. Pathogens often have different AT contents than the species being immunized, so altering the gene sequence of the immunogen to reflect the codons more commonly used in the target species may improve its expression.[13] Another consideration is the choice of promoter. The SV40 promoter was conventionally used until research showed that vectors driven by the Rous Sarcoma Virus (RSV) promoter had much higher expression rates.[2] More recently, expression rates have been further increased by the use of the cytomegalovirus (CMV) immediate early promoter. Inclusion of the Mason-Pfizer monkey virus (MPV)-CTE with/without rev increased envelope expression. Furthermore the CTE+rev construct was significantly more immunogenic then CTE alone vector. [14] Additional modifications to improve expression rates have included the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and modifications to the

polyadenylation and transcriptional termination sequences.[2] An example of DNA vaccine plasmid is pVAC, it uses SV40 promoter.
[edit]Vaccine

insert design

Immunogens can be targeted to various cellular compartments in order to improve antibody or cytotoxic T-cell responses. Secreted or plasma membrane-bound antigens are more effective at inducing antibody responses than cytosolic antigens, while cytotoxic T-cell responses can be improved by targeting antigens for cytoplasmic degradation and subsequent entry into the major histocompatibility complex (MHC) class I pathway.[1] This is usually accomplished by the addition of Nterminal ubiquitin signals.[15][16] The conformation of the protein can also have an effect on antibody responses, with ordered structures (like viral particles) being more effective than unordered structures.[17] Strings of minigenes (or MHC class I epitopes) from different pathogens are able to raise cytotoxic T-cell responses to a number of pathogens, especially if a TH epitope is also included.[1]
[edit]Delivery

methods

DNA vaccine and Gene therapy techniques are similar.

DNA vaccines have been introduced into animal tissues by a number of different methods. These delivery methods are briefly reviewed in Table 2, with the advantages and disadvantages of the most commonly used methods summarised in Table 3. The two most popular approaches are injection of DNA in saline, using a standard hypodermic needle, and gene gun delivery. A schematic outline of the construction of a DNA vaccine plasmid and its subsequent delivery by these two methods into a host is illustrated at Scientific American.[18] Injection in saline is normally conducted intramuscularly (IM) in skeletal muscle, or intradermally (ID), with DNA being delivered to the extracellular spaces. This can be assisted by electroporation[19]; by temporarily damaging muscle fibres with myotoxins such as bupivacaine; or by using hypertonic solutions of saline orsucrose.[2] Immune responses to this method of delivery can be

affected by many factors, including needle type,[8] needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected.[2] Gene gun delivery, the other commonly used method of delivery, ballistically accelerates plasmid DNA (pDNA) that has been adsorbed onto gold or tungstenmicroparticles into the target cells, using compressed helium as an accelerant.[2][12] Alternative delivery methods have included aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa,[12] and topical administration of pDNA to the eye[20] and vaginal mucosa.[12] Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations,[1]biodegradable microspheres,[21][12] attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa,[22] and recombinant adenovirus vectors.[12] The method of delivery determines the dose of DNA required to raise an effective immune response. Saline injections require variable amounts of DNA, from 10 g-1 mg, whereas gene gun deliveries require 100 to 1000 times less DNA than intramuscular saline injection to raise an effective immune response.[23] Generally, 0.2 g 20 g are required, although quantities as low as 16 ng have been reported.[2] These quantities vary from species to species, with mice, for example, requiring approximately 10 times less DNA than primates.[1] Saline injections require more DNA because the DNA is delivered to the extracellular spaces of the target tissue (normally muscle), where it has to overcome physical barriers (such as the basal lamina and large amounts of connective tissue, to mention a few) before it is taken up by the cells, while gene gun deliveries bombard DNA directly into the cells, resulting in less wastage. [1][2] Another approach to DNA vaccination is expression library immunization (ELI). Using this technique, potentially all the genes from a pathogen can be delivered at one time, which may be useful for pathogens which are difficult to attenuate or culture.[2] ELI can be used to identify which of the pathogens genes induce a protective response. This has been tested with Mycoplasma pulmonis, amurine lung pathogen with a relatively small genome, and it was found that even partial expression libraries can induce protection from subsequent challenge.[24]
Table 2. Summary of Plasmid DNA delivery methods

Method of Delivery

Formulation of DNA

Target Tissue

Amount of DNA

Injection (hypodermic needle)

Aqueous solution in saline

IM (skeletal); ID; (IV, subcutaneous and intraperitoneal with variable success)

Large amounts (approximately 100-200 g)

Parenteral Gene Gun DNA-coated gold beads

ED (abdominal skin); vaginal mucosa; Small amounts surgically exposed muscle (as little as 16 ng) and other organs

Pneumatic Aqueous solution (Jet) Injection

ED

Very high (as much as 300 g)

Topical application

Aqueous solution

Ocular; intravaginal

Small amounts (up to 100 g)

Cytofectin-mediated

Liposomes (cationic); microspheres; recombinant adenovirus vectors; attenuated Shigella vector; aerosolised cationic lipid formulations

IM; IV (to transfect tissues systemically); intraperitoneal; oral immunization to the intestinal mucosa; nasal/lung mucosal membranes

variable

Table 3. Advantages and disadvantages of commonly used DNA vaccine delivery methods

Method of Delivery Intramuscular or Intradermal injection

Advantage

Disadvantage

No special delivery mechanism Permanent or semi-permanent expression pDNA spreads rapidly throughout the body

Inefficient site for uptake due to morphology of muscle tissue

Relatively large amounts of DNA used Th1 response may not be the response required

Gene Gun

DNA bombarded directly into cells Small amounts DNA No particles required DNA can be delivered to cells mm to cm below skin surface

Th2 response may not be the response required Requires inert particles as carrier Significant shearing of DNA after high-pressure expulsion 10-fold lower expression, and lower immune response

Jet injection

Requires large amounts of DNA (up to 300 g)

High levels of immune response can be generated

Can increase transfection of intravenously delivered pDNA Intravenously delivered liposome-DNA complexes can potentially transfect all tissues

Liposome-mediated delivery

Toxicity Ineffectiveness in serum Risk of disease or immune reactions

Intranasally delivered liposome-DNA complexes can result in expression in distal mucosa as well as nasal muscosa and the generation of IgA antibodies

[edit]Immune [edit]Helper

response raised by DNA vaccines

T-Cell responses

DNA immunization is able to raise a range of TH responses, including lymphoproliferation and the generation of a variety of cytokine profiles. A major advantage of DNA vaccines is the ease with which they can be manipulated to bias the type of T-cell help towards a TH1 or TH2 response.[25] Each type of response has distinctive patterns of lymphokine and chemokine expression, specific types ofimmunoglobulins expressed, patterns of lymphocyte trafficking, and types of innate immune responses generated.
[edit]Raising of different types of T-cell help

The type of T-cell help raised is influenced by the method of delivery and the type of immunogen expressed, as well as the targeting of different lymphoid compartments.[2][26] Generally, saline needle injections (either IM or ID) tend to induce TH1 responses, while gene gun delivery raises TH2 responses.[25][26] This is true for intracellular and plasma membrane-bound antigens, but not for secreted antigens, which seem to generate TH2 responses, regardless of the method of delivery. [27] Generally the type of T-cell help raised is stable over time, and does not change when challenged or after subsequent immunizations which would normally have raised the opposite type of response in a nave animal.[25][26] However, Mor et al.. (1995)[9] immunized and boosted mice with pDNA encoding the circumsporozoite protein of the mouse malarial parasite Plasmodium yoelii (PyCSP) and found that the initial TH2 response changed, after boosting, to a TH1 response.

[edit]Mechanistic basis for different types of T-Cell help

It is not understood how these different methods of DNA immunization, or the forms of antigen expressed, raise different profiles of T-cell help. It was thought that the relatively large amounts of DNA used in IM injection were responsible for the induction of TH1 responses. However, evidence has shown no differences in TH type due to dose. [25] It has been postulated that the type of T-cell help raised is determined by the differentiated state of antigen presenting cells. Dendritic cells can differentiate to secrete IL-12 (which supports TH1 cell development) or IL-4 (which supports TH2 responses).[28] pDNA injected by needle is endocytosed into the dendritic cell, which is then stimulated to differentiate for TH1 cytokine production,[29] while the gene gun bombards the DNA directly into the cell, thus bypassing TH1 stimulation.
[edit]Practical uses of polarised T-Cell help

This polarisation in T-cell help is useful in influencing allergic responses and autoimmune diseases. In autoimmune diseases, the goal would be to shift the selfdestructive TH1 response (with its associated cytotoxic T cell activity) to a nondestructive TH2 response. This has been successfully applied in predisease priming for the desired type of response in preclinical models[1] and somewhat successful in shifting the response for an already established disease.[30]
[edit]Cytotoxic

T-cell responses

One of the greatest advantages of DNA vaccines is that they are able to induce cytotoxic T lymphocytes (CTL) without the inherent risk associated with live vaccines. CTL responses can be raised against immunodominant and immunorecessive CTL epitopes,[31] as well as subdominant CTL epitopes,[21] in a manner which appears to mimic natural infection. This may prove to be a useful tool in assessing CTL epitopes of an antigen, and their role in providing immunity. Cytotoxic T-cells recognise small peptides (8-10 amino acids) complexed to MHC class I molecules (Restifo et al., 1995). These peptides are derived from endogenous cytosolic proteins which are degraded and delivered to the nascent MHC class I molecule within the endoplasmic reticulum (ER).[32] Targeting gene products directly to the ER (by the addition of an amino-terminal insertionsequence) should thus enhance CTL responses. This has been successfully demonstrated using recombinant vaccinia viruses expressing influenza proteins,[32] but the principle should be applicable to DNA vaccines too. Targeting antigens for intracellular degradation (and thus entry into the MHC class I pathway) by the addition of ubiquitin signal sequences, or mutation of other signal sequences, has also been shown to be effective at increasing CTL responses.[16]

CTL responses can also be enhanced by co-inoculation with co-stimulatory molecules such as B7-1 or B7-2 for DNA vaccines against influenza nucleoprotein,[31][33] or GMCSF for DNA vaccines against the murine malaria model P. yoelii.[34] Co-inoculation with plasmids encoding co-stimulatory molecules IL-12 and TCA3 have also been shown to increase CTL activity against HIV-1 and influenza nucleoprotein antigens.[33][35]
[edit]Humoral

(antibody) response

Schematic diagram of an antibody and antigens

Antibody responses elicited by DNA vaccinations are influenced by a number of variables, including type of antigen encoded; location of expressed antigen (i.e. intracellular vs. secreted); number, frequency and dose of immunizations; site and method of antigen delivery, to name a few.
[edit]Kinetics of antibody response

Humoral responses after a single DNA injection can be much longer-lived than after a single injection with a recombinant protein. Antibody responses againsthepatitis B virus (HBV) envelope protein (HBsAg) have been sustained for up to 74 weeks without boost, while life-long maintenance of protective response to influenza haemagglutinin has been demonstrated in mice after gene gun delivery.[36] Antibody-secreting cells migrate to the bone marrow and spleen for long-term antibody production, and are generally localised there after one year.[36] Comparisons of antibody responses generated by natural (viral) infection, immunization with recombinant protein and immunization with pDNA are summarised in Table 4. DNAraised antibody responses rise much more slowly than when natural infection or

recombinant protein immunization occurs. It can take as long as 12 weeks to reach peak titres in mice, although boosting can increase the rate of antibody production. This slow response is probably due to the low levels of antigen expressed over several weeks, which supports both primary and secondary phases of antibody response.
Table 4. Comparison of T-Dependent Antibody Responses raise by DNA Immunizations, Protein Inoculations and Viral Infections

Method of Immunization

DNA Vaccine

Recombinant protein

Natural Infection

Amount of inducing antigen

ng

? (ng-g)

Duration of Ag presentation

several weeks

< 1 week

several weeks

Kinetics of Ab response

slow rise

rapid rise

rapid rise

Number of inoculations to obtain high avidity IgG and migration of ASC to bone marrow

one

two

one

Ab isotype (murine models)

C-dependent or Cindependent

C-dependent

Cindependent

Additionally, the titres of specific antibodies raised by DNA vaccination are lower than those obtained after vaccination with a recombinant protein. However, DNA immunization-induced antibodies show greater affinity to native epitopes than recombinant protein-induced antibodies. In other words, DNA immunization induces a qualitatively superior response. Antibody can be induced after just one vaccination with DNA, whereas recombinant protein vaccinations generally require a boost. As mentioned previously, DNA immunization can be used to bias the TH profile of the immune response, and thus the antibody isotype, which is not possible with either natural infection or recombinant protein immunization. Antibody responses generated by DNA are useful not just in vaccination but as a preparative tool, too. For example, polyclonal and monoclonal antibodies can be generated for use as reagents.
[edit]Mechanistic

basis for DNA raised immune responses

[edit]DNA

Uptake Mechanism

When DNA uptake and subsequent expression was first demonstrated in vivo in muscle cells,[37] it was thought that these cells were unique in this ability because of their extensive network of T-tubules. Using electron microscopy, it was proposed that DNA uptake was facilitated by caveolae (or, non-clathrin coated pits).[38] However, subsequent research revealed that other cells (such askeratinocytes, fibroblasts and epithelial Langerhans cells) could also internalize DNA.[30][39] This phenomenon has not been the subject of much research, so the actual mechanism of DNA uptake is not known. Two theories are currently popular that in vivo uptake of DNA occurs non-specifically, in a method similar to phago- or pinocytosis,[12] or through specific receptors.[40] These might include a 30kDa surface receptor, or macrophage scavenger receptors. The 30kDa surface receptor binds very specifically to 4500-bp genomic DNA fragments (which are then internalised) and is found on professional APCs and T-cells. Macrophage scavenger receptors bind to a variety of macromolecules, including polyribonucleotides, and are thus also candidates for DNA uptake.[40][41] Receptor mediated DNA uptake could be facilitated by the presence of polyguanylate sequences. Further research into this mechanism might seem pointless, considering that gene gun delivery systems, cationic liposome packaging, and other delivery methods bypass this entry method, but understanding it might be useful in reducing costs (e.g. by reducing the requirement for cytofectins), which will be important in the food animals industry.
[edit]Antigen

presentation by bone marrow-derived cells

A dendritic cell.

Studies using chimeric mice have shown that antigen is presented by bone-marrow derived cells, which include dendritic cells, macrophages and specialisedB-cells called professional antigen presenting cells (APC)[33][42] Iwasaki et al., 1997). After gene gun inoculation to the skin, transfected Langerhans cells migrate to the draining lymph node to present antigen.[1] After IM and ID injections, dendritic cells have also been

found to present antigen in the draining lymph node[39] and transfected macrophages have been found in the peripheral blood.[43] Besides direct transfection of dendritic cells or macrophages, cross priming is also known to occur following IM, ID and gene gun deliveries of DNA. Cross priming occurs when a bone marrow-derived cell presents peptides from proteins synthesised in another cell in the context of MHC class 1. This can prime cytotoxic T-cell responses and seems to be important for a full primary immune response.[1][44]
[edit]Role

of the target site

IM and ID delivery of DNA initiate immune responses differently. In the skin, keratinocytes, fibroblasts and Langerhans cells take up and express antigen, and are responsible for inducing a primary antibody response. Transfected Langerhans cells migrate out of the skin (within 12 hours) to the draining lymph node where they prime secondary B- and T-cell responses. In skeletal muscle, on the other hand, striated muscle cells are most frequently transfected, but seem to be unimportant in mounting an immune response. Instead, IM inoculated DNA washes into the draining lymph node within minutes, where distal dendritic cells are transfected and then initiate an immune response. Transfected myocytes seem to act as a reservoir of antigen for trafficking professional APCs.[12][37][44]
[edit]Maintenance

of immune response

DNA vaccination generates an effective immune memory via the display of antigenantibody complexes on follicular dendritic cells (FDC), which are potent B-cell stimulators. T-cells can be stimulated by similar, germinal centre dendritic cells. FDC are able to generate an immune memory because antibodies production overlaps longterm expression of antigen, allowing antigen-antibody immunocomplexes to form and be displayed by FDC.[1]
[edit]Interferons

Both helper and cytotoxic T-cells can control viral infections by secreting interferons. Cytotoxic T cells usually kill virally infected cells. However, they can also be stimulated to secrete antiviral cytokines such as INF- and TNF-, which dont kill the cell but place severe limitations on viral infection by down-regulating the expression of viral components.[45] DNA vaccinations can thus be used to curb viral infections by nondestructive IFN-mediated control. This has been demonstrated for the hepatitis B virus.[46] IFN- is also critically important in controlling malaria infections,[47] and should be taken into consideration when developing anti-malarial DNA vaccines.
[edit]Modulation

of the immune response

[edit]Cytokine

modulation

For a vaccine to be effective, it must induce an appropriate immune response for a given pathogen, and the ability of DNA vaccines to polarise T-cell help towards TH1 or TH2 profiles, and generate CTL and/or antibody when required, is a great advantage in this regard. This can be accomplished by modifications to the form of antigen expressed (i.e. intracellular vs. secreted), the method and route of delivery, and the dose of DNA delivered.[25][26][48][49][50] However, it can also be accomplished by the co-administration of plasmid DNA encoding immune regulatory molecules, i.e. cytokines, lymphokines or costimulatory molecules. These genetic adjuvants can be administered a number of ways:

as a mixture of 2 separate plasmids, one encoding the immunogen and the other encoding the cytokine; as a single bi- or polycistronic vector, separated by spacer regions; or as a plasmid-encoded chimera, or fusion protein.

In general, co-administration of pro-inflammatory agents (such as various interleukins, tumor necrosis factor, and GM-CSF) plus TH2 inducing cytokines increase antibody responses, whereas pro-inflammatory agents and TH1 inducing cytokines decrease humoral responses and increase cytotoxic responses (which is more important in viral protection, for example). Co-stimulatory molecules like B7-1, B7-2 and CD40L are also sometimes used. This concept has been successfully applied in topical administration of pDNA encoding IL-10.[20] Plasmid encoded B7-1 (a ligand on APCs) has successfully enhanced the immune response in anti-tumour models, and mixing plasmids encoding GM-CSF and the circumsporozoite protein of P. yoelii (PyCSP) has enhanced protection against subsequent challenge (whereas plasmid-encoded PyCSP alone did not). It was proposed that GM-CSF may cause dendritic cells to present antigen more efficiently, and enhance IL-2 production and TH cell activation, thus driving the increased immune response.[34] This can be further enhanced by first priming with a pPyCSP and pGM-CSF mixture, and later boosting with a recombinant poxvirus expressing PyCSP. [51] However, co-injection of plasmids encoding GM-CSF (or IFN-, or IL-2) and a fusion protein of P. chabaudi merozoite surface protein 1 (C-terminus)-hepatitis B virus surface protein (PcMSP1-HBs) actually abolished protection against challenge, compared to protection acquired by delivery of pPcMSP1-HBs alone.[17] The advantages of using genetic adjuvants are their low cost and simplicity of administration, as well as avoidance of unstable recombinant cytokines and potentially toxic, conventional adjuvants (such as alum, calcium phosphate, monophosphoryl lipid

A, cholera toxin, cationic and mannan-coated liposomes, QS21, carboxymethylcellulose and ubenimix).[1][12] However, the potential toxicity of prolonged cytokine expression has not been established, and in many commercially important animal species, cytokine genes still need to be identified and isolated. In addition, various plasmid encoded cytokines modulate the immune system differently according to the time of delivery. For example, some cytokine plasmid DNAs are best delivered after the immunogen pDNA, because pre- or co-delivery can actually decrease specific responses, and increase nonspecific responses.[52]
[edit]Immunostimulatory

CpG motifs

Plasmid DNA itself appears to have an adjuvant effect on the immune system.[1][2] Bacterially derived DNA has been found to trigger innate immune defence mechanisms, the activation of dendritic cells, and the production of TH1 cytokines.[29][53] This is due to recognition of certain CpG dinucleotide sequences which are immunostimulatory.[49][54] CpG stimulatory (CpG-S) sequences occur twenty times more frequently in bacterially derived DNA than in eukaryotes. This is because eukaryotes exhibit CpG suppression i.e. CpG dinucleotide pairs occur much less frequently than expected. Additionally, CpG-S sequences are hypomethylated. This occurs frequently in bacterial DNA, while CpG motifs occurring in eukaryotes are all methylated at the cytosine nucleotide. In contrast, nucleotide sequences which inhibit the activation of an immune response (termed CpG neutralising, or CpG-N) are over represented in eukaryotic genomes.[55] The optimal immunostimulatory sequence has been found to be an unmethylated CpG dinucleotide flanked by two 5 purines and two 3 pyrimidines.[49][53] Additionally, flanking regions outside this immunostimulatory hexamer must be guanine-rich to ensure binding and uptake into target cells. The innate system works synergistically with the adaptive immune system to mount a response against the DNA encoded protein. CpG-S sequences induce polyclonal B-cell activation and the upregulation of cytokine expression and secretion.[56] Stimulated macrophages secrete IL-12, IL-18, TNF-, IFN-, IFN- and IFN-, while stimulated Bcells secrete IL-6 and some IL-12.[12][56][57] Manipulation of CpG-S and CpG-N sequences in the plasmid backbone of DNA vaccines can ensure the success of the immune response to the encoded antigen, and drive the immune response toward a TH1 phenotype. This is useful if a pathogen requires a TH response for protection. CpG-S sequences have also been used as external adjuvants for both DNA and recombinant protein vaccination with variable success rates. Other organisms with hypomethylated CpG motifs have also demonstrated the stimulation of polyclonal B-cell expansion. However, the mechanism

behind this may be more complicated than simple methylation hypomethylated murine DNA has not been found to mount an immune response. Most of the evidence for the existence of immunostimulatory CpG sequences comes from murine studies. Clearly, extrapolation of this data to other species should be done with caution different species may require different flanking sequences, as binding specificities of scavenger receptors differ between species. Additionally, species such as ruminants may be insensitive to immunostimulatory sequences due to the large gastrointestinal load they exhibit. Further research may be useful in the optimisation of DNA vaccination, especially in the food animal industry.
[edit]Alternative

boosts

DNA-primed immune responses can be boosted by the administration of recombinant protein or recombinant poxviruses. Prime-boost strategies with recombinant protein have successfully increased both neutralising antibody titre, and antibody avidity and persistence, for weak immunogens, such as HIV-1 envelope protein.[1][58] Recombinant virus boosts have been shown to be very efficient at boosting DNA-primed CTL responses. Priming with DNA focuses the immune response on the required immunogen, while boosting with the recombinant virus provides a larger amount of expressed antigen, leading to a large increase in specific CTL responses. Prime-boost strategies have been successful in inducing protection against malarial challenge in a number of studies. Primed mice with plasmid DNA encoding Plasmodium yoelii circumsporozoite surface protein (PyCSP), then boosted with a recombinant vaccinia virus expressing the same protein had significantly higher levels of antibody, CTL activity and IFN-, and hence higher levels of protection, than mice immunized and boosted with plasmid DNA alone.[59] This can be further enhanced by priming with a mixture of plasmids encoding PyCSP and murine GM-CSF, before boosting with recombinant vaccinia virus.[51] An effective prime-boost strategy for the simian malarial model P. knowlesi has also been demonstrated.[60] Rhesus monkeys were primed with a multicomponent, multistage DNA vaccine encoding two liver-stage antigens - the circumsporozoite surface protein (PkCSP) and sporozoite surface protein 2 (PkSSP2) and two blood stage antigens - the apical merozoite surface protein 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42). They were then boosted with a recombinant canarypox virus encoding all four antigens (ALVAC-4). Immunized monkeys developed antibodies against sporozoites and infected erythrocytes, and IFN--secreting T-cell responses against peptides from PkCSP. Partial protection against sporozoite challenge was achieved, and mean parasitemia was significantly reduced, compared to control monkeys. These models, while not ideal for extrapolation to P. falciparum in humans, will be important in pre-clinical trials.

[edit]Additional

methods of enhancing DNA-Raised immune responses

[edit]Formulations of DNA

The efficiency of DNA immunization can be improved by stabilising DNA against degradation, and increasing the efficiency of delivery of DNA into antigen presenting cells.[1] This has been demonstrated by coating biodegradable cationic microparticles (such as poly(lactide-co-glycolide) formulated with cetyltrimethylammonium bromide) with DNA. Such DNA-coated microparticles can be as effective at raising CTL as recombinant vaccinia viruses, especially when mixed with alum. Particles 300 nm in diameter appear to be most efficient for uptake by antigen presenting cells. [1]
[edit]Alphavirus vectors

Recombinant alphavirus-based vectors have also been used to improve DNA vaccination efficiency.[1] The gene encoding the antigen of interest is inserted into the alphavirus replicon, replacing structural genes but leaving non-structural replicase genes intact. The Sindbis virus and Semliki Forest virus have been used to build recombinant alphavirus replicons. Unlike conventional DNA vaccinations, however, alphavirus vectors kill transfected cells, and are only transiently expressed. Also, alphavirus replicase genes are expressed in addition to the vaccine insert. It is not clear how alphavirus replicons raise an immune response, but it is thought that this may be due to the high levels of protein expressed by this vector, replicon-induced cytokine responses, or replicon-induced apoptosis leading to enhanced antigen uptake by dendritic cells.
[edit]

DNA Vaccines

Genetic/ DNA immunization is a novel technique used to efficiently stimulate humoral and cellular immune responses to protein antigens. The direct injection of genetic material into a living host causes a small amount of its cells to produce the introduced gene products. This inappropriate gene expression within the host has important immunological consequences, resulting in the specific immune activation of the host against the gene delivered antigen (Koprowski et al, 1998).

Traditional Vaccines: The development of vaccination against harmful pathogenic microorganisms represents an important advancement in the history of modern medicine. In the past, traditional vaccination has relied on two specific types of microbiological preparations to produce material for immunization and generation of a protective immune response. These two categories involve either living infectious material that has been manufactured in a weaker state and therefore inhibits the vaccine from causing disease, or inert, inactivated, or subunit preparations.
Immunization

Live attenuated vaccines stimulate protective immune responses when they replicate in the host. The viral proteins produced within the host are released into the extracellular space surrounding the infected cells and are then acquired, internalized and digested by scavenger cells that circulate the body. These cells are called antigen presenting cells (APC s) and inclu de macr ophages, dendritic cells, and B cells, which work together to expand immune response. The APCs recirculate fragments of the digested the antigen to their surface, attached to MHC class IIantigens. This complex of foreign peptide antigen plus host MHC class II antigens forms part of the specific signal with which APCs along with the MHC peptide complex, trigger the action of of immune cells, the T helper lymphocytes. The second part of the activation signal comes from the APCs themselves, which display on their cell surface constimulatory molecules along with MHC-antigen complexes. Both drive T call expansion and activation through interaction with their respective ligands, the T cell receptor complex (TCR) and the constimulatory receptors CD28/CTLA4, present on the the T cell surface. Activated T cells secrete molecules that act as powerful activates of immune cells. Also as viral proteins are produced within the host cells, small parts of these proteins surface, chaperoned by host cell MHC class Iantigens. These complexes together are recognized by a second class of T cells, killer or cytotoxic cells. This recognition, along with other stimulation by APCs and production of cytokine by

stimulated T cells, is responsible for the developments of mature cytotoxic T cells (CTL) capable of destroying infected cells. In most instances live infection induces life long immunity. Although live attenuated preparations are the vaccines of choice they do pose the risk of reversion to their pathogenic form, causing infection.
Immune Response

In contrast, when inoculated nonlive vaccines composed of whole or even partial viruses are not produced within the host cells, they mainly end up in the extracellular space. They provide protection by directly generating T helper and humoral immune responses against the pathogenic immunogen. In the absence of the cellular production of the foreign antigen, these vaccines are usually devoid of the ability to induce significant T cytotoxic responses. In addition, these vaccines are not actually produced in the host, and therefore, they are not customized by the host. The immunity induced by their vaccines frequently decreases during the life of the host and may require additional boosters to achieve lifelong immunity. However, nonlive vaccines offer some important advantages over live vaccines: they are produced earlier, and they can be designed to contain only the specific antigenic target of the pathogen that is involved in the development of protective immunity and exclude all other viral components.

Genetic Immunization: Since its early applications in the 1950's, DNA-based immunization has become a novel approach to vaccine development. Direct injection of naked plasmid DNA induces strong immune responses to the antigen encoded by the gene vaccine. Once the plasmid DNA construct is injected the host cells take up the foreign DNA, expressing the viral gene and producng the corresponding viral protein inside the cell. This form of antigen presentation and processing induced both MHC and class I and class II restricted cellular and humoral immune responses (Encke, J. et al, 1999).

History: The use of genetic material to deliver genes for therapeutic purposes has been practiced for many years. Experiments outlining the transfer of DNA into cells of living animals were reported as early as 1950. Later experiments using purified genetic material only further confirmed that the direct DNA gene injection in the absence of viral vectors results in the expression of the inoculated genes in the host. There have been additional experiments that extend these findings to recombinant DNA molecules, further illustrating the idea that purified nucleic acids could be directly delivered into a host and proteins would be produced. In 1992, scientists Tang and Johnson reported that the delivery of human growth hormone in a expression cassette in vivo resulted in production of detectable levels of the growth hormone in host mice. They also found that these inoculated mice developed antibodies against the human growth hormone; they termed this immunization procedure "genetic immunization", which describes the ability of inoculated genes to be individual immunogens (Koprowski et al, 1998).
DNA Vaccines

Construction: DNA vaccines are composed of a bacterial plasmids. Expression plasmids used in DNA-based vaccination normally contain two unites: the antigen expression unit composed of promoter/enhancer sequences, followed by antigen-encoding and polyadenylation sequences and the production unit composed of of bacterial sequences necessary for plamid amplification and selection (Schirmbeck, R., 2001). The construction of bacterial plasmids with vaccine inserts is accomplished using recombinant DNA technology. Once constructed, the vaccine plasmid is transformed into bacteria, where bacterial growth produces multiple plasmid copies. The plasmid DNA is then purified from the bacteria, by separating the circular plasmid from the much larger bacterial DNA and other bacterial impurities. This purifies DNA acts as the vaccine (AAM, 1996).

DNA vaccine plasmid

Administration- Over the past decade of clinical research and trials, several possible routs of plasmid delivery have been found. Successful immunization has been demonstrated after delivery of plasmids through intramuscular, intradermal and intravenous injection. The skin and mucous membranes being considered the best site for immunization due to the high concentrations of dendritic cells (DC), macrophages and lymphocytes (Raz,E., 1998). Intradermal injection of DNA-coated gold particles with a gene gun have been used. The plasmid DNA can be diluted in distilled water, saline or sucrose. There has also been positive demonstration of proinjection or codelivery with various drugs (Encke et al,1999). Mechanisms: A plasmid vector that expresses the protein of interest (e.g. viral protein) under the control of an appropriate promoter is injected into the skin or muscle of the the host. After uptake of the plasmid, the protein is produced endogenously and intracellularly processed into small antigenic peptides by the host proteases. The peptides then enter the lumen of the endoplasmic reticulum (E.R.) by membrane-associated transporters. In the E.R., peptides bind to MHC class I molecules. These peptides are presented on the cell surface in the context of the MHC class I. Subsequent CD8+ cytotoxic T cells (CTL) are

stimulated and they evoke cell-mediated immunity. CTLs inhibit viruses through both cytolysis of infected cells and noncytolysis mechanisms such as cytokine production (Encke et al, 1999). The foreign protein can also be presented by the MHC class II pathway by APCs which elicit helper T cells (CD4+) responses. These CD4+ cells are able to recognize the peptides formed from exogenous proteins that were endocytosed or phagocytosed by APC, then degraded to peptide fragments and loaded onto MHC class II molecules. Depending on the the type of CD4+ cell that binds to the complex, B cells are stimulated and antibody production is stimulated. This is the same manner in which traditional vaccines work (Schirmbeck et al., 2001).
DNA Vaccine Mechanism

Advantages: DNA immunization offers many advantages over the traditional forms of vaccination. It is able to induce the expression of antigens that resemble native viral epitopes more closely than standard vaccines do since live attenuated and killed vaccines are often altered in their protein structure and antigenicity. Plasmid vectors can be constructed and produced quickly and the coding sequence can be manipulated in many ways. DNA vaccines encoding several antigens or proteins can be delivered to the host in a single dose, only requiring a microgram of plasmids to induce immune responses. Rapid and large-scale production are available at costs considerably lower than traditional vaccines, and they are also very temperature stable making storage and transport much easier. Another important advantage of genetic vaccines is their therapeutic potential for ongoing chronic viral infections. DNA vaccination may provide an important tool for stimulating an immune response in HBV, HCV and HIV patients. The continuos expression of the viral antigen caused by gene vaccination in an environment containing many APCs may promote successful therapeutic immune response which cannot be obtained by other traditional vaccines (Encke et al, 1999). This is a subject that has generated a lot of interest in the last five years. Limitations: Although DNA can be used to raise immune responses against pathogenic proteins, certain microbes have outer capsids that are made up of polysaccharides. This limits the extent of the usage of DNA vaccines because they cannot substitute for polysaccharide-based subunit vaccines (AMM, 1996). Future- It has recently been discovered that the transfection of myocytes can be amplified by pretreatment with local anesthetics or with cardiotoxin,

which induce local tissue damage and initiate myoblast regeneration. Gaining a full understanding of this mechanism of DNA uptake could prove helpful in improving applications for gene therapy and gene vaccination. Both improved expression and better engineering of the DNA plasmid may enhance antibody response to the gene products and expand the applications of the gene vaccines (Raz, E., 1998).

DNA Probes Disease diagnosis is an important feature in animalhealth care. Diagnostic tests are currently being developed for the following purposes. 1. to detect infectious diseases 2. to detect specific forms of cancer 3. to detect AIDS viruses in blood for transfusion 4. to detect genetic disorder 5. to detect high- and low-density lipoproteins which are indicative of a possible heart disease, etc. Diagnostic tests are mainly of 2 types: 1. those using monoclonal antibodies 2. those using DNA probes The role of monoclonal the previous chapter. antibodies in disease diagnosis has been discussed in detail in

Although DNA probes have direct competition from diagnostics using

monoclonal antibodies,

DNA probes will probably be more effective for detecting bacterial infections and genetic disorders. Further, while monoclonal antibodies are non-proprietary,

DNA probes can be protected by patents and this offers the producers potentially larger shares of any market they penetrate.

Antisense Nucleotides as Therapeutic AgentsA very effective and specific approach for treatment of a variety of diseases is to design and use oligonucleotides (say 25-35 bases long) complementary to the 5f-ends of the parasite mRNAs; such oligonucleotides are called antisense oligonucleotides. The antisense oligonucleotide may be linked to an acridine for increased effectiveness. When such oligonucleotides were used on cultured blood parasite Trypanosoma brucei, the parasite was killed. This approach has been quite successful in the treatment of cancer, and antisense oligonucleotides are in various stages of evaluation.

Monoclonal

Antibodies-

A monoclonal antibody (Mab) preparation is specific to a single antigenic determinant (epitope) of a single antigen. Mabs are being produced against a variety of antigens and being employed for many purposes including disease diagnosis. Mabs are employed for (i) (ii) classification clear and of blood specific groups detection (ABO, of Rh etc.),

pathogens,

(iii) and a very early and accurate detection of cancers. (ii) After 20 yeas, in June 1992, United Nations Conference on Environment and Development (UNCED), popularly described as Rio Earth Summit, was help in Rio de Janeiro (Brazil), where heads of the states from 166 countries participated to examine the issues involved and the solutions possible. (iii) The latest conference, the World Summit on Sustainable Development (WSSD) was held in Johannesburg (South Africa) during August 26 to September 4, 2002 to assess global change since the above Rio Earth Summit. While on the one hand, there is an increasing problem of the conservation of nature and natural resources. Both these problems are receiving constant attention of environmentalists. Among implications, there is also an alarm due to release of genetically engineered organisms in the atmosphere and due to release of effluents from biotechnological companies, so that the environmentalists are having a debate on the effects of developments in biotechnology on the environment. There is also a debate on the safety of the use of the products of biotechnology, an area described as biosafety. Among application, on the other hand, efforts are also being made to use biotechnology to protect the environment from pollution and to conserve natural resources. At a time, when the gap between those who have plenty and those who do not have even the minimum is widening, both ends of this spectrum, i.e. plenty and poverty, are contributing to environmental degradation. It is, therefore, necessary that the developing and developed countries jointly find a path of development which meets the needs of the present without compromising the ability, of future generations to meet their needs (World Commission on Environment and Development). Efforts are being made to achieve this objective through a variety of approaches, and biotechnology is certainly one of them. In this chapter and the next four chapters, environmental; implications and applications of biotechnology for environment will be discussed. In recent years, we have witnessed a debate on the environmental implications of biotechnology. In this debate, risks involved in the use of biotechnological approaches have often been emphasized (or even overemphasized) and the adequate guidelines for safety have been suggested ad enforced by law. However, there have also been rapid developments in the applications of biotechnology, which may help in controlling environment pollution, thus giving a cleaner and sustainable environment in future. According to one estimate in USA, the US market for environmental clean-up applications was expected to grow at an average rate of 17%, while that for microbes and enzymes was expected to grow by only 7% every year. Besides others, these applications for environment clean-up include biotreatment methods for effluents and toxic wastes (this

subject is described asbioremediation and is discussed in the next). However, these bioremediation treatments, it is feared, could be problematic, where they involve deliberate or accidental release of genetically modified microbes to the environment. These applications of biotechnology in environment management.

What research is being done on Huntington's disease?

Although Huntington's disease attracted considerable attention from scientists in the early 20th century, there was little sustained research on the disease until the late 1960s when the Committee to Combat Huntington's Disease and the Huntington's Chorea Foundation, later called the Hereditary Disease Foundation, first began to fund research and to campaign for federal funding. In 1977, Congress established the Commission for the Control of Huntington's Disease and Its Consequences, which made a series of important recommendations. Since then, Congress has provided consistent support for federal research, primarily through the National Institute of Neurological Disorders and Stroke, the government's lead agency for biomedical research on disorders of the brain and nervous system. The effort to combat Huntington's disease proceeds along the following lines of inquiry, each providing important information about the disease: Basic neurobiology. Now that the Huntington's disease gene has been located, investigators in the field of neurobiology-which encompasses the anatomy, physiology, and biochemistry of the nervous system-are continuing to study the Huntington's disease gene with an eye toward understanding how it causes disease in the human body. Clinical research. Neurologists, psychologists, psychiatrists, and other investigators are improving our understanding of the symptoms and progression of the disease in patients while attempting to develop new therapeutics. Imaging. Scientific investigations using PET and other technologies are enabling scientists to see what the defective gene does to various structures in the brain and how it affects the body's chemistry and metabolism. Animal models. Laboratory animals, such as mice, are being bred in the hope of duplicating the clinical features of Huntington's disease and can soon be expected to help scientists learn more about the symptoms and progression of the disease. Fetal tissue research. Investigators are implanting fetal tissue in rodents and nonhuman primates with the hope that success in this area will lead to understanding, restoring, or replacing functions typically lost by neuronal degeneration in individuals with Huntington's disease. These areas of research are slowly converging and, in the process, are yielding important clues about the gene's relentless destruction of mind and body. The NINDS supports much of this exciting work. Molecular Genetics

For 10 years, scientists focused on a segment of chromosome 4 and, in 1993, finally isolated the Huntington's disease gene. The process of isolating the responsible genemotivated by the desire to find a curewas more difficult than anticipated. Scientists now believe that identifying the location of the Huntington's disease gene is the first step on the road to a cure. Finding the Huntington's disease gene involved an intense molecular genetics research effort with cooperating investigators from around the globe. In early 1993, the collaborating scientists announced they had isolated the unstable triplet repeat DNA sequence that has the Huntington's disease gene. Investigators relied on the NINDS-supported Research Roster for Huntington's Disease, based at Indiana University in Indianapolis, to accomplish this work. First started in 1979, the roster contains data on many American families with Huntington's disease, provides statistical and demographic data to scientists, and serves as a liaison between investigators and specific families. It provided the DNA from many families affected by Huntington's disease to investigators involved in the search for the gene and was an important component in the identification of Huntington's disease markers. For several years, NINDS-supported investigators involved in the search for the Huntington's disease gene made yearly visits to the largest known kindred with Huntington's disease14,000 individuals who live on Lake Maracaibo in Venezuela. The continuing trips enable scientists to study inheritance patterns of several interrelated families. The Huntington's disease Gene and Its Product Although scientists know that certain brain cells die in Huntington's disease, the cause of their death is still unknown. Recessive diseases are usually thought to result from a gene that fails to produce adequate amounts of a substance essential to normal function. This is known as a loss-of-function gene. Some dominantly inherited disorders, such as Huntington's disease, are thought to involve a gene that actively interferes with the normal function of the cell. This is known as a gain-of-function gene. How does the defective Huntington's disease gene cause harm? The Huntington's disease gene encodes a proteinwhich has been named huntingtinthe function of which is as yet unknown. The repeated CAG sequence in the gene causes an abnormal form of huntingtin to be made, in which the amino acid glutamine is repeated. It is the presence of this abnormal form, and not the absence of the normal form, that causes harm in Huntington's disease. This explains why the disease is dominant and why two copies of the defective geneone from both the mother and the fatherdo not cause a more serious case than inheritance from only one parent. With the Huntington's disease gene isolated, NINDS-supported investigators are now turning their attention toward discovering the normal function of huntingtin and how the altered form causes harm. Scientists hope to reproduce, study, and correct these changes in animal models of the disease. Huntingtin is found everywhere in the body but only outside the cell's nucleus. Mice called "knockout mice" are bred in the laboratory to produce no huntingtin; they fail to develop past a very early embryo stage and quickly die. Huntingtin, scientists now know, is necessary for life. Investigators hope to learn why the abnormal version of the protein damages only certain parts of the brain. One theory is that cells in these parts of the brain may be supersensitive to this abnormal protein. Cell Death in Huntington's disease Although the precise cause of cell death in Huntington's disease is not yet known, scientists are paying close attention to the process of genetically programmed cell death that occurs deep within the brains of individuals with Huntington's disease. This process involves a complex series of interlinked events leading to cellular suicide. Related areas of investigation include: Excitotoxicity. Overstimulation of cells by natural chemicals found in the brain.

Defective energy metabolism. A defect in the power plant of the cell, called mitochondria, where energy is produced. Oxidative stress. Normal metabolic activity in the brain that produces toxic compounds called free radicals. Trophic factors. Natural chemical substances found in the human body that may protect against cell death. Several Huntington's disease studies are aimed at understanding losses of nerve cells and receptors in Huntington's disease. Neurons in the striatum are classified both by their size (large, medium, or small) and appearance (spiny or aspiny). Each type of neuron contains combinations of neurotransmitters. Scientists know that the destructive process of Huntington's disease affects different subsets of neurons to varying degrees. The hallmark of Huntington's disease, they are learning, is selective degeneration of medium-sized spiny neurons in the striatum. NINDS-supported studies also suggest that losses of certain types of neurons and receptors are responsible for different symptoms and stages of Huntington's disease. What do these changes look like? In spiny neurons, investigators have observed two types of changes, each affecting the nerve cells' dendrites. Dendrites, found on every nerve cell, extend out from the cell body and are responsible for receiving messages from other nerve cells. In the intermediate stages of Huntington's disease, dendrites grow out of control. New, incomplete branches form and other branches become contorted. In advanced, severe stages of Huntington's disease, degenerative changes cause sections of dendrites to swell, break off, or disappear altogether. Investigators believe that these alterations may be an attempt by the cell to rebuild nerve cell contacts lost early in the disease. As the new dendrites establish connections, however, they may in fact contribute to nerve cell death. Such studies give compelling, visible evidence of the progressive nature of Huntington's disease and suggest that new experimental therapies must consider the state of cellular degeneration. Scientists do not yet know exactly how these changes affect subsets of nerve cells outside the striatum. Animal Models of Huntington's disease As more is learned about cellular degeneration in Huntington's disease, investigators hope to reproduce these changes in animal models and to find a way to correct or halt the process of nerve cell death. Such models serve the scientific community in general by providing a means to test the safety of new classes of drugs in nonhuman primates. NINDS-supported scientists are currently working to develop both nonhuman primate and mouse models to investigate nerve degeneration in Huntington's disease and to study the effects of excitotoxicity on nerve cells in the brain. Investigators are working to build genetic models of Huntington's disease using transgenic mice. To do this, scientists transfer the altered human Huntington's disease gene into mouse embryos so that the animals will develop the anatomical and biological characteristics of Huntington's disease. This genetic model of mouse Huntington's disease will enable in-depth study of the disease and testing of new therapeutic compounds. Another idea is to insert into mice a section of DNA containing CAG repeats in the abnormal, disease gene range. This mouse equivalent of Huntington's disease could allow scientists to explore the basis of CAG instability and its role in the disease process. Fetal Tissue Research A relatively new field in biomedical research involves the use of brain tissue grafts to study, and potentially treat, neurodegenerative disorders. In this technique, tissue that has degenerated is replaced with implants of fresh, fetal tissue, taken at the very early stages of development. Investigators are interested in applying brain tissue implants to Huntington's disease research.

Extensive animal studies will be required to learn if this technique could be of value in patients with Huntington's disease. Clinical Studies Scientists are pursuing clinical studies that may one day lead to the development of new drugs or other treatments to halt the disease's progression. Examples of NINDS-supported investigations, using both asymptomatic and symptomatic individuals, include: Genetic studies on age of onset, inheritance patterns, and markers found within families. These studies may shed additional light on how Huntington's disease is passed from generation to generation. Studies of cognition, intelligence, and movement. Studies of abnormal eye movements, both horizontal and vertical, and tests of patients' skills in a number of learning, memory, neuropsychological, and motor tasks may serve to identify when the various symptoms of Huntington's disease appear and to characterize their range and severity. Clinical trials of drugs. Testing of various drugs may lead to new treatments and at the same time improve our understanding of the disease process in Huntington's disease. Classes of drugs being tested include those that control symptoms, slow the rate of progression of Huntington's disease, and block effects of excitotoxins, and those that might correct or replace other metabolic defects contributing to the development and progression of Huntington's disease. Imaging NINDS-supported scientists are using positron emission tomography (PET) to learn how the gene affects the chemical systems of the body. PET visualizes metabolic or chemical abnormalities in the body, and investigators hope to ascertain if PET scans can reveal any abnormalities that signal Huntington's disease. Investigators conducting Huntington's disease research are also using PET to characterize neurons that have died and chemicals that are depleted in parts of the brain affected by Huntington's disease. Like PET, a form of magnetic resonance imaging (MRI) called functional MRI can measure increases or decreases in certain brain chemicals thought to play a key role in Huntington's disease. Functional MRI studies are also helping investigators understand how Huntington's disease kills neurons in different regions of the brain. Imaging technologies allow investigators to view changes in the volume and structures of the brain and to pinpoint when these changes occur in Huntington's disease. Scientists know that in brains affected by Huntington's disease, the basal ganglia, cortex, and ventricles all show atrophy or other alterations.

Gene Therapy & Huntington's Disease

By Jenn Foreacre, eHow Contributor

While there is no cure for the degenerative brain disease known as Huntington's Disease, there are some treatment options that can make life more comfortable for the patient and his family. Medications are currently used to treat the effects of Huntington's Disease, but gene therapy is being researched in the hopes that it may be able to restore brain function and perhaps reverse the effects of the disease.

What is Huntington's Disease?

1. Huntington's Disease, or HD, is a brain disease that causes the degeneration of genetically programed neurons. Degeneration occurs in certain areas of the brain, particularly in those areas

that control intelligence, emotional responses and movement. The age of onset and progression of Huntington's Disease can vary from one person to another.

Causes & Symptoms

2. The disease is typically passed on from parent to child via a mutation of a normal gene. Approximately 30,000 people in the United States are affected by Huntington's Disease, and children of HD parents have a 50 percent chance of inheriting the HD gene. Inheriting the gene means that an individual will develop the disease sooner or later. Early symptoms of Huntington's Disease are depression, mood swing, irritability, and trouble with driving, remembering facts, learning new things and making decisions. As the disease takes hold and progresses, the ability to concentrate on an intellectual task increases in difficulty. An HD person may also have difficulty with swallowing and feeding herself. As of October 2009, there is no cure for Huntington's Disease. There is no way to reverse the course of it or stop it entirely. However, medication and gene therapy can help to control many of the problems associated with the disease.

What is Gene Therapy?

3. Gene therapy is the process of inserting specific genes into a individual's cells in order to treat a disease. Gene therapy is typically used to treat hereditary diseases, such as Huntington's Disease. Its purpose is to provide a defective or degenerated gene with alternative genes that are still fully functional. As of October 2009, gene therapy is still in the very early stages of development, though it has shown success with some hereditary diseases.

Treating Huntington's Disease with Gene Therapy

4. Gene therapy is appealing for Huntington's Disease patients because of its potential to replace dying neurons with healthy ones, thus improving and even restoring brain function. Animal and human pilot studies have shown success with the use of embryonic stem cells. In the studies, transplanted cells not only survived, but flourished in their new environment by growing and establishing connections with other neurons. However, the use of human embryonic cells is controversial, and raises ethical questions. It is suggested by some that HD researchers will need to find another source of neuronal cells.

Other Treatment Options

5. While gene therapy continues to be researched, physicians and medical teams are using medication to help manage the effects of Huntington's Disease. Because depression, delusions, violent outbursts and mood swings are common side effects of the disease, anti-psychotic drugs, anti-depressants, tranquilizers and mood stabilizers are used. These types of medications often have significant side effects, so most physicians prescribe the lowest possible dose. Because HD patients can have trouble with meals due to movement problems, difficulty swallowing and jaw clenching, it is recommended that food be cut into small pieces, or softened or pureed. Patients should avoid dairy products because of their tendency to increase mucus secretions. Vitamins and nutritional supplements are recommended for patients who are having difficulty consuming enough calories. As the disease progresses, bendable straws for drinking may be necessary, and feeding tubes might also need to be considered. Because there is no cure for Huntington's Disease, it will continue to progress, and will usually run its full course in 10 to 30 years. In the final stages of the disease a patient will often become

bedridden, and will most likely die from heart failure, pneumonia or a related complication. (See Reference 4)

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Advances In The Treatment Of Parkinson's Disease Jill Marjama-Lyons, M.D.

Jill Marjama-Lyons, M.D. is an Assistant Professor Department of Neurology, Shands Jacksonville and Director of The Parkinson and Movement Disorder Center.

Historical Perspective
Since James Parkinson's publication on The Shaking Palsy in 1817, the treatment of Parkinson's disease has undergone several transformations. Over a century elapsed devoid of any effective treatments for this devastating disease and Parkinson's disease was considered to be a terminal illness. In the 1940's and 1950's neurosurgical treatments of Parkinson's disease emerged. Neurosurgery of the basal ganglia was performed as early as 1939 by Meyers with notable improvement in the motor symptoms of Parkinson's disease, but with a high mortality rate of 12%.1 The development of the stereotactic frame in 1947 allowed for more precise lesioning of the brain and over tens of thousands of thalamotomies and pallidotomies were performed in Europe and the United States for the treatment of Parkinson's disease.2,3 Pharmacotherapy during the 1940's and 1950's consisted primarily of the use of amantadine and cogentin.4,5 These drugs reduced the motor symptoms of Parkinson's disease, but were clearly less efficacious as the disease progressed. The 1967 discovery of levodopa by Cotzias revolutionized the treatment of Parkinson's disease.6 The dramatic improvement in the motor symptoms of Parkinson's disease with carbidopa/levodopa (Sinemet) gave birth to the levodopa era and a near complete halt to the neuro-surgical treatments of the prior two decades. Introduction of the dopamine agonists, bromocriptine (Parlodel) and pergolide (Permax) occurred in the 1970's. The dopamine agonists were clearly beneficial in reducing tremor, rigidity and bradykinesia, but were less efficacious and had a greater side effect profile than carbidopa/levodopa and were primarily were used as adjunctive therapy to carbidopa/levodopa.7,8 The addition of selegiline and controlled release carbidopa/levodopa (Sinemet CR) in the early 1990's had minimal impact on clinical treatment of Parkinson's disease. Today, levodopa remains the gold standard for the treatment of Parkinson's disease. However, long-term use of levodopa over 5 to 10 years is often associated with the development of motor complications in as high as 80% of Parkinson patients.9,10,11 These

include wearing off of the levodopa dose prior to taking the next dose(requiring frequent dosing of the medication sometimes as often as every 1 to 2 hours throughout the day), dose failures, rapid changes in motor symptoms so called "on/off" phenomena, and disabling dyskinesias (involuntary chorea and dystonia). The limitations of long-term levodopa therapy has led to the study and development of alternative and adjunctive medications for the treatment of Parkinson's disease. In the last two years, four new medications have been FDA approved and are currently available for the treatment of Parkinson's patients. These include two dopamine agonists, pramipexole (Mirapex) and ropinerole (Requip) and two comt (catecholamine-o-methyl transferase) inhibitors, tolcapone (Tasmar) and entacapone (Comtan). A brief description and potential uses of these medications will be described below. In addition to the development of new medications, a renewed interest in the neurosurgical treatment of Parkinson's disease has emerged in the last decade. An understanding of the neuroanatomy and physiology of the basal ganglia coupled with recent advances in the technology of stereotactic surgery has led to effective neurosurgical procedures for Parkinson's disease. Some of these techniques will be discussed later in this article.

New Medications For Parkinson's Disease

Dopamine Agonists The role of dopamine agonists as solely adjunctive medications to levodopa is changing. Recent studies of the new dopamine agonists clearly demonstrate they are effective in reducing the motor symptoms (tremor, rigidity, bradykinesia) of Parkinson's disease as monotherapy as well as when added to levodopa.12,13 Off time decreases by 30% to56%, less on/off fluctuations occur and a recent 5 year study showed a markedly lower incidence of dyskinesia in the patients on monotherapy ropinerole of 5% when compared to the monotherapy levodopa rate of 46%.14 Similar data has been reported in MPTP-treated monkeys for bromocriptine and ropinerole.15, 16 There is also evidence that dopamine agonists may be neuroprotective of dopaminergic neurons.17-20 Taken together, these findings support the use of dopamine agonists as monotherapy in newly diagnosed, early and mild to moderate Parkinson's disease and adding levodopa therapy when the patients motor symptoms are not adequately controlled by dopamine agonists alone or intolerable side effects develop. Four dopamine agonists are available in the United States and include bromocriptine, pergolide, mirapex and ropinerole. The new, second generation agonists, mirapex andropinerole are structurally more similar to the dopamine molecule, lack the ergot structure of the first generation agonists, are potent D-2 agonists similar to pergolide, and have little or no binding to alpha, beta, and serotonin receptors. Despite these differences, no one dopamine agonist has been shown to be superior to another in the treatment of the motor symptoms of Parkinson's disease.21,22 They all may produce similar side effects of nausea/vomiting, light headedness, orthostasis, peripheral edema, sedation and confusion/hallucinations. The ergot agonists have rarely been reported to cause retroperitoneal and pulmonary fibrosis, erythromelalgia and Raynaud's like syndrome.23 In the authors experience ropinerole has a lower CNS side effect profile and maybe a more appropriate agonist for an elderly Parkinson's patient with preexisting confusion or

dementia. Recent reports in the literature have implicated sudden sleep attacks in Parkinson's patients taking mirapex and in only one patient taking ropinerole with some of these attacks occurring while the patient was driving.24 Given all of the above factors, it may be difficult to decide which dopamine agonist to use in a Parkinson's patient. Some general rules to consider are outlined below: 1. Use only one dopamine agonist at a time as monotherapy or adjunctive to levodopa; 2. Begin with the lowest dose (bromocriptine 2.5mg, pergolide 0.05mg, mirapex 0.125mg, ropinerole 0.25mg); 3. Always dose three times daily evenly spaced throughout the day; 4. Slowly titrate the dosage by doubling the lowest dose no faster than every 1-2 weeks until an intolerable side effect occurs or clinical benefit is achieved. Be aware of the potency differences among the different agonists ( pergolide and mirapex are roughly equivalent; for example 0.5mg of pergolide could be substituted for 0.5mg of mirapex, bromocriptine is 1/10 as potent as pergolide and mirapex such that 5mg of bromocriptine could be substituted for 0.5mg of pergolide and mirapex, and ropinerole is more potent than bromocriptine but less than pergolide and mirapex and may need to be given at single doses of 4-6mg to achieve a clinical benefit. 5. If a patient has preexisting dementia or confusion, consider using ropinerole. COMT Inhibitors COMT inhibitors act by inhibiting the enzyme catecholamine-o-methyltransferase which is responsible for metabolizing levodopa to3-O-methyldopa in the peripheral bloodstream. By inhibiting COMT, these drugs result in an increase in the half-life of levodopa and an increase in the bioavailability of levodopa such that a more continuous and greater amount of levodopa crosses the blood brain barrier to act directly on dopaminergic neurons. Tolcapone was released in March of 1998 and Entacapone was just released in November of 1999. Both result in a decrease in the "off" time by 1 to 3 hours per day, a decrease in motor fluctuations and allowed for a reduction in the total levodopa daily dose by 10 to 30%.25-28 COMT inhibitors are indicated for use as adjunctive therapy to levodopa in Parkinson's disease patients who experience wearing "off" and motor fluctuations. Both drugs may cause levodopa side effects of dyskinesias, nausea, confusion and sedation that are easily controlled by lowering the levodopa dose. Non levodopa side effects include diarrhea (3 to 4%) and urine discoloration. Tolcapone results in an increase in liver enzymes in only 1 to 3% of patients. Three cases of fatal liver failure out of 60,000 patient years have been reported with the use of tolcapone and the FDA has mandated that patients on tolcapone have frequent liver function testing.29 Patients taking tolcapone must have their liver function checked every 2 weeks for the first year, followed by one a month for the next 6 months and then every 2 months there-after. No cases of elevated liver enzymes or hepatotoxicity have been reported with entacapone to date. Dosing of the COMT inhibitors varies between the two drugs. Tolcapone should be started at 100 mg three times a day separated by 6 to 8 hours and if no significant benefit is noted by 2 to 3 weeks one should either discontinue the drug or consider increasing the dosage to 200 mg t.i.d. If however at the higher dosage no significant clinical improvement is observed the drug should be stopped. Entacapone comes only in 200 mg pills and it is recommended that the patient take one pill each time they take levodopa up to a maximum

of 8 total pills per day. As with tolcapone if no clinical benefit is noted one should consider stopping the drug. Due to the relatively little time entacapone has been available it is difficult to comment on it's true clinical impact on the treatment of Parkinson's disease. Tolcapone, on the other hand, has been used worldwide for over a year and a half and in the author's personal experience, it is clearly miraculous in a select group of patients suffering from motor fluctuations. Three patients I personally treated had such severe motor fluctuations that all three were candidates for neurosurgical treatments and all three dramatically improved with the addition of tolcapone such that we were able to lower their levodopa doses and avoid pursuing neurosurgical therapies.

Neurosurgical Procedures
A greater understanding of the neuroanatomy and physiology of the basal ganglia coupled with recent advances in the technology of stereotactic surgery has led to effective and safer neurosurgical procedures for Parkinson's disease. These include tissue transplants (human and porcein fetal midbrain), direct lesioning of the brain (pallidotomy, thalamotomy) and placement of an electrode deep into the brain and then stimulating the brain with high frequencies (deep brain stimulation). Due to the limited scope of this article, a description of the most effective and widely used procedures of pallidotomy and deep brain stimulation will follow. Pallidotomy Pallidotomy involves placing a small lesion into the medial segment of the globus pallidus with thermocoagulation. Despite its long history, patients undergoing pallidotomy have not been studied carefully until recently. Current data suggests that patients may experience a reduction in rigidity, bradykinesia, off time, dyskinesia and tremor up to 50% which may persist between 6 months and 4 years.25-27 Mortality is less than 1%, a significant improvement from the 12% rate of the 1950's. Morbidity occurs in 1 to 8% of patients and may include hemiparesis, visual field defects, depression, hypophonia, dysarthria, and seizures. Bilateral pallidotomy is generally not recommended due to the higher morbidity rate. Deep Brain Stimulation Deep brain stimulation involves placing a small quadripolar electrode into the brain at a specific site and then continuously stimulating the brain at frequencies between 100 and 180 hertz. This technique has been applied to the ventral intermediate nucleus (VIM) of the thalamus, the subthalamic nucleus (STN) and the globus pallidus intermedius (GPI). Stimulation of the VIM causes a significant reduction in tremor in 92% of Parkinson's disease patients that persists past 8 years.28 However, VIM stimulation does not result in marked reduction of rigidity or bradykinesia. Recent studies with STN stimulation have reported an 80% reduction in the Unified Parkinson's Disease Rating Scale (UPDRS) tremor score as well as a 65% reduction in rigidity and a 51% reduction in bradykinesia. Similar, although less dramatic, findings occur with deep brain stimulation of the GPI.2931 Deep brain stimulation when compared to ablative procedures, has a much lower morbidity rate especially with bilateral procedures. Overall morbidity has been estimated at 2% for permanent neurological deficits and minor side effects are often reversible with electrode reprogramming. Current studies comparing pallidotomy to deep brain stimulation

are underway in an effort to decide if one of these procedures is more effective in the treatment of Parkinson's disease and which patients seem to benefit most. Deciding to refer a Parkinson's patient for neurosurgical treatment may be a difficult task. Neurosurgery is generally recommended for patients who have severe motor fluctuations, disabling dyskinesia, and are classified as a stage III or higher on the Hoehn and Yahr scale. Evaluation for a neurosurgical procedure should be done at a center with a neurologist who specializes in Parkinson's disease and a neurosurgeon with stereotactic training.

Conclusion
The spectrum of treatments for Parkinson's disease has dramatically changed in the last decade. The advances in pharmacotherapy and neurosurgery afford today's practicing neurologist a greater opportunity to better manage the motor symptoms of Parkinson's patients and allow for a higher quality of daily life in those suffering from this sometimes cruel and unpredictable disease. As we enter the 21st century, future research will undoubtedly lead to more effective treatments and new discoveries in the area of neuroprotective agents and restorative cell transplants (neurostem cells) may eventually lead to a cure for Parkinson's disease.
REFERENCES

Major Development In Treatment Of Huntington's Disease

(Ivanhoe Newswire) -- Huntington's disease is incurable, but scientists believe they have a major development in the treatment of the disease. Scientists at the Buck Institute for Age Research have discovered that a family of enzymes is new targets for the treatment of Huntington's disease (HD). HD is a progressive neurodegenerative genetic disorder which causes uncontrolled movements, cognitive difficulties, emotional disturbances, and dementia. The signs of the disease begin in middle age and affect about 30,000 people in the U.S. If a parent has the disease there is a 50% chance that the child will develop it. The victims of HD will live 15-25 years after the symptoms erupt, and be incapacitated prior to death. HD is caused by a mutation in the Huntington gene located on the chromosome number four. The mutation causes abnormalities in the Huntington protein (mHTT). The pathology of HD is sped up when mHTT is sliced into smaller, incredibly toxic fragments. Capases, a family of intracellular that mediate cell death, and calpains, enzymes regulated by the concentration of calcium ions, are closely associated with the slicing of these fragments.

The study used proteases, enzymes that speed up the breakdown of protein in reaction to water. This is the first time all 514 proteases in humans were individually studied in cell culture to see how the affect the mHTT slicing. The Buck Institute researchers found 11 proteases that when inhibited reduce the slicing of mHTT, in turn reducing the toxic fragments associated with HD. Four of the proteases belong to the MMP family. The MMP enzyme was verified in mice with HD, and homologs of MMP inhibited HDinduced neuronal dysfunction in fruit flies. Dr. Lisa Ellerby, member of the Buck Institute research team was quoted as saying "we'll also be crossing mice that no longer have particular MMPs with those who have HD to see what effect that has on offspring." The researchers have pinpointed a place to start searching for the treatment of HD, which could be very beneficial to treatment options in the future, and the possible curing or deceleration of HD.
Augmentation therapy is strategy that is being explored as a way to improve the odds of relieving OCD symptoms when treating patients with OCD medication. Augmentation therapy involves using combinations of drug, rather than a single OCD medication, for maximum effect. Augmentation strategies could be especially effective for people who do not respond to standard OCD medication. Why Augmentation Therapy? If you have OCD, you may know that a variety of treatments are available. However, you may also know that not all people respond to these treatments. Although the introduction of selective serotonin reuptake inhibitors (SSRIs)such as Luvox (Fluvoxamine), Prozac (Fluoxetine),Paxil (Paroxetine) and Zoloft (Sertraline), and tricyclic antidepressants such as Anafranil (Clompiramine) have been a huge step forward in the treatment of OCD, 40% to 60% of people will not respond adequately to these drugs. As in other areas of medicine, psychiatrists are now exploring whether treatment of OCD with a combination of medications, rather than a single medication, offers more relief, for more people. Antipsychotic Medications are Used to Augment Current Treatments Although antidepressants are the standard medical treatment for OCD, it has been suggested that adding antipsychotic drugs to a treatment plan could be helpful in improving OCD symptoms. Why is this? First, antipsychotic medications such as Risperdal (Respiridone), Zyprexa (Olanzapine) or Seroquel (Quetiapine) affect levels of the neurotransmitter dopamine. Problems with the dopamine system have been implicated in OCD. In addition, some people with OCD have difficulty believing that their obsessions and/orcompulsions are illogical or unreasonable. A failure to recognize that obsessions and/or compulsions do not make sense has been shown to be a barrier to benefiting from standard treatments. It has been suggested that antipsychotic medications may be effective in helping change this pattern of thinking. Does Augmentation Therapy Work? In general, the available scientific evidence strongly supports the use of antipsychotic medications as useful augmentating drugs for adults whose OCD symptoms have not responded to standard treatments. However, you must keep in mind that there are two categories of antipsychotic medications, each with their own potential side effects. The first generation or "typical" antipsychotics tend to have side effects related to abnormal movements such as tardive dyskinesia, which involves involuntary and uncontrollable movement of different parts of the body including the mouth and face. Tardive dyskinesia can sometimes be permanent if not addressed promptly. The second generation or "atypical" antipsychotics usually have less risk of tardive dyskinesia, but can cause metabolic problems such as weight gain and elevated blood sugars and cholesterol.

Given this, the potential benefits of using an antipsychotic medication as an augmentation strategy for reducing OCD symptoms should outweigh the risks. In this respect, the relatively less severe side effects of the second generation or atypical antipsychotics often make them a first choice as augmentation agents. As with any medical treatment, the decision to add an antipsychotic medication to your current treatment plan is a choice that should be made in strong collaboration with your family doctor or psychiatrist.
Neural transplantation studies where foetal striatal tissue is grafted into the striatum of patients with Huntingtons disease have taken place at several sites worldwide in recent years, following success in rodent models of the disease. Studies have for the most part been safe but have had various degrees of effectiveness. This article looks at the successes and failures of these studies and considers what has been learnt in terms of safety, techniques and methodology. While knowledge of the optimal protocol is advancing, there are still many aspects that need refining, such as immunosuppression and grafting technique. Although advances in this field are hampered by the need for more complete knowledge of the disease itself, the future of neural transplantation has a great deal of potential.

ancer can be described as a disease where cellular communication has broken down, allowing transformed cells to escape tight regulatory signals and to replicate autonomously and continously, ultimately invading and interfering with the functions of normal tissues. Under physiological conditions cells communicate with one another by activating receptors at the cell surface, which convey the signal through pathways of proteins located in the cytoplasm and subsequently through networks of transcription factors in the nucleus with control the expression of genes that mediate the cell's response. In this article we briefly describe the gene therapy approaches that have been adopted in an effort to treat cancer. Typically, cancer develops as a result of aberrant growth factor signalling, where a pathway that instructs a cell to grow and divide becomes constitutively active. This arises through mutations in a growth factor receptor, or through mutations in the components of cell signalling pathways. (Collectively, genes that encode for mutated cellular proteins involved in promoting cell growth are termed oncogenes). Threrefore, in order to effectively treat a cancer, it is essential that all cells that carry a mutated oncogene, are destroyed, otherwise the cancer will continue to grow and spread.

Gene Therapy Approaches

There are a number of gene therapy-based clinical trials in the field of oncology currently underway worldwide, encompassing a variety of delivery agents, target indications and means of intervention. All therapeutic strategies that are being developed in the treatment of cancer ultimately aim to destroy cancerous cells whilst leaving normal cells unaffected. This can be achieved by using one (or more) of the following approaches: i. Expression of tumour suppressor genes In many types of cancer, deactivating mutations arise in specific genes whose function is to prevent the uncontrolled growth of the cell. Genes that regulate cell division in this manner are called tumour suppressor genes (TSG); two examples include p53 and Rb. It has been found that re-introduction this type of gene into cancer cells initiates a series of events that results in the ultimate death of the cancer cell. Moreover, because normal cells of the body have the correct copy of these genes, the expression of a TSG in normal cells has no significant pathological effects. Therefore, it is generally considered that TSG expression in tumours is a safe way to specifically kill the cells that comprise the cancer. The only state-approved gene therapy treatment adopts this type of approach and is used in the treatment of both lung cancer and squamous cancer of the head and neck. This drug, called gendicine (gene medicine) and comprises an adenovirus expressing p53, was registered by the Chinese State Food and Drug Administration (SFDA) in 2003 and has been successfully used in the treatment of these two cancers, specifically when administered in conjunction with traditionally used chemotherapeutics.

ii. Oncolytic viruses For almost a century it has been known that certain types of virus can kill cancer cells if they are induced to replicate inside them. Research is currently being conducted by institutions around the world using both "non engineered" and "engineered" viruses to evaluate their use in the fight against multiple types of cancer. Non engineered viruses are naturally occurring viruses that innately preferentially target and replicate in certain types of tumor cells. Some non-engineered viruses include the Newcastle Disease Virus, Autonomous Parvovirus, and the Reovirus. Conversely, engineered viruses do not innately selectively target and replicate in cancer cells. Scientists must genetically modify ("engineer") the virus to selectively target and/or replicate within specific types of cancer cells. Today, there are three main approaches that are being explored in the development of engineered tumor-specific oncolytic viruses. Although the three approaches differ from one another, they all share a common goalthe destruction of cancer cells as a result of viral replication. The three approaches are as follows: 1. Selective TargetingCapsid Protein Modification: The capsid protein, the external surface of the virus, is modified so that the virus will specifically target cancer cells, completely avoiding normal cells. The virus would then replicate within the targeted cancer cell, ultimately leading to cell death. 2. Selective Replication in the Absence of an Antitumor Gene: The virus is genetically modified so that it will replicate only in the absence of a gene believed to inhibit tumor cell growth, such as P53. While the virus "passes through" normal cells, it is triggered to replicate in cancer cells that do not exhibit an antitumor gene, ultimately leading to cancer cell death. 3. Selective Replication in the Presence of Unique Tumor Cell Characteristic: The virus is genetically modified so that it will replicate only in the presence of a characteristic (e.g. an antigen) unique to the specific type of cancer. While the virus passes through normal cells, it is triggered to replicate in cancer cells that exhibit a specific characteristic, ultimately leading to cancer cell death. Cell Genesys' oncolytic virus product platform utilizes this approach. Research and development efforts are currently under way at numerous organizations to thoroughly evaluate the safety and efficacy of oncolytic virus therapies. The majority of the studies currently being conducted are utilizing intratumoral delivery of the oncolytic virus therapies. Direct administration into the tumor allows the oncolytic virus immediate "access" to the cancer cells in which they replicate and ultimately destroy. Additional studies are being conducted to determine the safety and efficacy of intravenous administration of oncolytic virus therapy. While intravenous administration holds the promise of providing systemic treatment and targeting cells that have spread from the primary tumor source, researchers are faced with the challenge of maintaining therapeutic levels of the virus in the presence of the immune system which intrinsically seeks to rid viruses from the body. Intravenously administered oncolytic virus therapy must overcome pre-existing antibodies in order to achieve therapeutic effect. Alternatively, research is being conducted to determine a way to modulate the immune response allowing the virus time to reach the primary tumor source as well as target cancer cells that have spread throughout the body. iii. Expression of genes that initiate cell death Programmed cell death, known as apoptosis, is an essential cellular homeostasis mechanism that ensures correct development and function of multi-cellular organisms. The pivotal importance of correct execution of apoptosis is apparent from the many human diseases with aberrancies in apoptosis, including cancer. During cancer development, various imbalances can arise in the apoptotic machinery. Consequently, sensitivity towards apoptosis is progressively reduced, which ultimately leads to inappropriate cell survival and malignant progression. However, it has become clear that cancer cells are often reliant on these aberrancies for continued survival. Perhaps counter intuitively, cancer cells can in fact be more prone to apoptosis than normal cells. The apoptosis-prone phenotype of cancer cells is masked and counterbalanced by upregulation of one or more antiapoptotic mechanisms. Therefore, it is of enormous therapeutic interest to selectively tip the balance of the cellular fate of cancer cells towards apoptosis. Gene therapy can be employed to exploit this weakness in cancer cells by using recombinant gene transfer vectors to express genes that promote programmed cell death. These can either be the ligands that activate death receptor signalling, or the components of the apoptotic machinery that mediate these signals. At present there are a wealth of clinical trials underway that exploit the sensitivity of cancer cells to pro-apoptotic signals and it represents an emerging sub-field within the gene therapy arena.

iv. Gene-directed enzyme pro-drug therapy In this approach tumour cells are killed by the specific action of a toxic drug that is produced from a non-toxic precursor by the action of an activating enzyme. In the clinical setting, an appropriate vector (typically viral) is first used to achieve expression of a prodrug-activating enzyme inside tumour cells. A nontoxic (or minimally toxic)

prodrug is then administered and is converted to the active, toxic metabolite in cells expressing the activating enzyme. These cells are subsequently killed. Moreover, untransduced cells might also be killed following prodrug activation, by mechanisms that include direct transfer of activated drug through gap junctions, ingestion of apoptotic bodies from killed cells, effects on tumour vasculature, or immunological responses. v. Immunotherapy Immunotherapy is used to stimulate the body's immune system against cancer. This can be achieved in many different ways. One example is the use of vaccines composed of antigens derived from tumor cells to boost the body's production of antibodies or immune cells (T lymphocytes) to fight the cancer.Typically in this approach, gene transfer vectors are used to deliver cancer antigens to the immune cells, either by engineering the patients own T lymphocytes out of their body and re-administrating them, or by simple injection of the gene transfer vectors encoding the antigens so that the patients immune cells become infected in vivo. In a second example, a patients anti-tumour immune response can be boosted by the administration of cytokines (natural chemicals in our body that control our immune system) or antibodies that function to specifically attack cancer cells and stimulate the immune system to destroying the tumour. Immunotherapy is a huge field in its own right and is outwith the scope of this website. Readers can find more information on this topic by following the following link. In general, genetic disorders are inherited diseases that arise when someone has two dysfunctional copies of a single gene on both of their chromosomes. The result of this is often devastating, resulting in abnormal functioning of specific cells and the development of an often life-threatening condition. In these cases gene therapy is employed to replace the dysfunctional gene with a correct copy and restore the normal functioning of affected cells. In this page we describe some of the most well-known genetic disorders that are being targeted by the gene therapy community.

1. Cystic Fibrosis
Cystic fibrosis (CF) is an inherited disease of your mucus and sweat glands. It affects mostly your lungs, pancreas, liver, intestines, sinuses, and sex organs. Normally, mucus is watery. It keeps the linings of certain organs moist and prevents them from drying out or getting infected. But in CF, an abnormal gene (CFTR) causes mucus to become thick and sticky. The mucus builds up in your lungs and blocks the airways. This makes it easy for bacteria to grow and leads to repeated serious lung infections. Over time, these infections can cause serious damage to your lungs. The thick, sticky mucus can also block tubes, or ducts, in your pancreas. As a result, digestive enzymes that are produced by your pancreas cannot reach your small intestine. These enzymes help break down the food that you eat. Without them, your intestines cannot absorb fats and proteins fully. The abnormal gene also causes your sweat to become extremely salty. As a result, when you perspire, your body loses large amounts of salt. This can upset the balance of minerals in your blood. The imbalance may cause you to have a heat emergency. CF can also cause infertility (mostly in men). The symptoms and severity of CF vary from person to person. Some people with CF have serious lung and digestive problems. Other people have more mild disease that doesn't show up until they are adolescents or young adults. Respiratory failure is the most common cause of death in people with CF. Until the 1980s, most deaths from CF occurred in children and teenagers. Today, with improved treatments, people with CF live, on average, to be more than 35 years old

Gene Therapy Approaches

Only a year after the gene for CF was identified scientists showed in cells in the laboratory that it was possible to correct the CF defect by adding a healthy copy of the gene. These findings have been consistently repeated by other groups using a variety of models and gene transfer agents. Studies in humans followed quickly. Gene therapy for CF has been tested in humans using both viruses and liposomes. These early studies were concerned mainly with safety issues. The amount of gene transfer achieved is similar for both systems and is probably still too small to have any real therapeutic benefit - although it is important to note that none of these trials actually measured therapeutic benefit. Also at present the effect seen (gene expression) only lasts for a few days. The scientific principle of gene therapy for CF is a sound one and the technology already exists to transfer the CF gene into the airway cells in man but there are still challenges ahead of the field. The two main challenges are getting the gene into the cells more efficiently and to make gene expression last longer. However the field is moving forward rapidly and with the development of better new viruses and liposomes these problems are surmountable. After initial encouraging clinical trials most research for CF gene therapy has returned to the laboratory to solve the problems mentioned above.

2. SCID
The body's immune system fights against diseases and infections. The SCID syndromes are inherited disorders that result in severe defects in the immune system. White blood cells (which fight infection) are produced in the

bone marrow by stem cells. In people with SCID, the bone marrow stem cells are absent or defective. This leaves the affected person open to any and all germs around him because he has no way to fight them off. There are several different types of SCID. The most common type is called X-linked SCID because its gene is linked to the X chromosome that a mother provides to her child during reproduction. To break down the genetics simply, males are more likely to develop this type of SCID because they only have one X chromosome--the traits tied to it are more likely to be expressed. Females, on the other hand, have two X chromosomes -- both must carry the SCID gene in order for a girl to have the disease. The second most common type comes from the lack of the enzyme adenosine deaminase. The disorder is inherited when both parents contribute an abnormal gene to a child during reproduction. This is referred to as autosomal recessive inheritance. Since a child is born with SCID, the diagnosis is usually made before age 6 months. The child has a high number of infections that are more serious and less responsive to antibiotics than normal. These infections may be ones that are unusual for a child to have, such as Pneumocystis carinii pneumonia (PCP). The child may have persistent diarrhea with weight loss and chronic skin infections. Since 1968, the treatment of choice has been replacing the defective bone marrow with normal bone marrow through stem cell (bone marrow) transplant. This is a difficult treatment that does not guarantee a cure, but it does work in many cases. Children with SCID are usually kept isolated from other people, including relatives, and take Bactrim (trimethoprim/sulfamethoxazole) to prevent PCP infection. Without a bone marrow transplant, the child is always at risk for a severe or fatal infection and will have difficulty surviving beyond his first birthday.

Gene Therapy Approaches

In order to treat a child using gene therapy s ome of the child's own marrow stem cells are removed. The "normal" version of the defective gene responsible for SCID (the gamma-receptor) is inserted into the cells. The "corrected" cells are then put back into the child's bone marrow. Gene therapy for SCID has been tried successfully. On April 3, 2002, physicians in England announced that they had successfully restored the immune system of a boy with X-linked SCID. Eighteen-month-old Rhys Evans appeared to have been "cured" of the disorder, although whether one treatment will last a lifetime is not yet known. Other children have also been treated successfully. A complication developed with gene therapy trials in France, however, beginning in September 2002. Unfortunately, three boys treated as infants later developed a leukemia-like blood disorder. They have been treated with chemotherapy. Researchers are working on discovering why this happened, and how to ensure that other children with SCID who are treated enjoy the success of gene therapy without the negative consequences. Based on the French cases, the U.S. Food and Drug Administration (FDA) suspended two similar American studies.

3. Muscular Dystrophy
Muscular dystrophy (MD) is a genetic disorder that weakens the muscles that help the body move. People with MD have incorrect or missing information in their genes, which prevents them from making the proteins they need for healthy muscles. Because MD is genetic, people are born with the problem - it's not contagious and you can't catch it from someone who has it. MD weakens muscles over time, so children, teens, and adults who have the disease can gradually lose the ability to do the things most people take for granted, like walking or sitting up. Someone with MD might start having muscle problems as a baby or their symptoms might start later. Some people even develop MD as adults. There are several major forms of muscular dystrophy that affect teens, each of which weakens different muscle groups in various ways.

Duchenne (pronounced: due-shen) muscular dystrophy (DMD), the most common type of the disease, is caused by a problem with the gene that makes a protein calleddystrophin. This protein helps muscle cells keep

their shape and strength. Without it, muscles break down and a person gradually becomes weaker. DMD affects boys. Symptoms usually start between ages 2 and 6. By age 10 or 12, kids with DMD often need to use a wheelchair. The heart may also be affected, and people with DMD need to be followed closely by a lung and heart specialist. They can also develop scoliosis (curvature of the spine) and tightness in their joints. Over time, even the muscles that control breathing get weaker, and a person might need a ventilator to breathe. People with DMD usually do not survive beyond their late teens or early adulthood. Becker muscular dystrophy (BMD), like DMD, affects boys. The disease is very similar to DMD, but its symptoms may start later and can be less severe. With BMD, symptoms like muscle breakdown and weakness sometimes don't begin until age 10 or even in adulthood. People with BMD can also have breathing, heart, bone, muscle, and joint problems. Many people with BMD can live long, active lives without using a wheelchair. How long a person with BMD can live varies depending on the severity of any breathing and heart problems. Emery-Dreifuss (pronounced: em-uh-ree dry-fuss) muscular dystrophy (EDMD)typically starts causing symptoms in late childhood to early teens and sometimes as late as age 25. EDMD is another form of muscular dystrophy that affects mostly boys. It involves muscles in the shoulders, upper arms, and shins, and it often causes joint problems (joints can become tighter in people with EDMD). The heart muscle may also be affected. Limb-girdle muscular dystrophy (LGMD) affects boys and girls equally, weakening muscles in the shoulders and upper arms and around the hips and thighs. LGMD can begin as early as childhood or as late as midadulthood, and it often progresses slowly. Over time, a wheelchair might be necessary to get around. There are many different types of LGMD, each with its own specific features. Facioscapulohumeral (pronounced: fa-she-o-skap-you-lo-hyoo-meh-rul) muscular dystrophy (FSHD) can affect both guys and girls, and it usually begins during the teens or early adulthood. FSHD affects muscles in the face and shoulders and sometimes causes weakness in the lower legs. People with this type of MD might have trouble raising their arms, whistling, or tightly closing their eyes. How much a person with this form of muscular dystrophy is affected by the condition varies from person to person. It can be quite mild in some people. Myotonic (pronounced: my-uh-tah-nick) dystrophy (MMD) is a form of muscular dystrophy in which the muscles have difficulty relaxing. In teens, it can cause a number of problems, including muscle weakness and wasting (where the muscles shrink over time), cataracts, and heart problems. Congenital muscular dystrophy (CMD) is the term for all types of MD that show signs in babies and young children, although the MD isn't always diagnosed right away. Like other forms of MD, CMD involves muscle weakness and poor muscle tone. Occurring in both girls and boys, it can have different symptoms. It varies in how severely it affects people and how quickly or slowly it worsens. In rare cases, CMD can cause learning disabilities or mental retardation. The life expectancy (in other words, how long a person may live) for many of these forms of muscular dystrophy depends on the degree to which a person's muscles are weakened as well as how much the heart and lungs are affected.

Gene Therapy Approaches

Traditionally it was thought that standard gene transfer technology would be best applicable to the treatment of this disease, i.e. the use of gene transfer vectors to deliver a corrected copy of the dysfunctional gene in muscle cells. In this vein, a substantial amount of work has gone into the construction of non-viral and viral systems that deliver various versions of the genes mutated in the variety of dystrophies. In the case of Duchenne's Muscular Dystrophy, where the mutated gene is a few megabases in length, a huge plethora of vectors have been constructed that express shorter functional versions of the full-length dystrophin protein. It has now been realised, however, that this type of replacement gene therapy is not the only way to treat this devastating disease. Recent research has shown that it is possible to 'correct' a patients own copy of the defective genes by a process known as exon skipping. Exon skipping is achievable for the treatment of DMD due to the repetitive nature of the gene and it has proved possible to prevent expression of the mutated protein by allowing the cell's trancription machinery to skip the mutation that causes the disease to form a slightly shorter functional version of the full-length protein. In this approach it is not strictly speaking the gene that is corrected, but the messenger that actually produces the protein from the DNA code. This approach is now feasible because of a new chemical means of synthesising the short nucleic acids that are required to induce the exon skipping. Using modification to the natural nucleic acids it is possible to synthesise oligonucleotides based on morpholino chemistry to induce exon skipping. These morpholinos are more stable in patients and can enter more muscle cells than traditional oligonucleotides. This imporved technology is now under evaluation in the clinic and the first results from these trials should provide some insight into the potential success of this approach.

Gene Therapy of Infectious Disease

Written by webmaster Friday, 05 September 2008 15:43

Gene therapy can also be used to treat life-threatening infections where no other treatment is available. In this case, genetic information is specifically introduced into infected cells with a view to prevent the effective replication of the target pathogen. In this section we describe some of the approaches that have been adopted in the treatment of the most prevalent infectious diseases.
1. AIDS/HIV

HIV (human immunodeficiency virus) is the virus that causes AIDS (acquired immunodeficiency syndrome). HIV destroys certain white blood cells called CD4+ T cells. These cells are critical to the normal function of the human immune system, which defends the body against illness. When HIV weakens the immune system, a person is more susceptible to developing a variety of cancers and becoming infected with viruses, bacteria and parasites. It is for this reason that HIV infections are ultimately fatal, as the body can no longer fight off infections that it normally can deal with quite easily.
2. Hepatitis/HBV

One of the major causes of hepatitis is infection with the Hepatitis B virus (HBV). HBV itself does not directly cause damage to the liver. Rather, the body's immune response to the virus paradoxically causes the damage. Thus, in an HBV infection, the body's immune response to the virus is responsible for both the elimination of the virus from the body and recovery from the infection. Yet, at the same time, the injury to the liver cells is caused by that same immune response. Hepatitis B virus is spread or acquired through exposure to infected blood or the body's secretions. The highest concentrations of hepatitis B virus are found in the blood, semen, vaginal discharge, breast milk, and saliva. About one third of the world's population has been exposed at some time to HBV. Moreover, approximately 350 million individuals worldwide are chronically (long duration) infected with this virus. As a result, the complications of HBV infection lead to two million deaths annually.
3. Gene Therapy Approaches

The gene therapy approaches used to treat both HIV and HBV are much the same, as the ultimate aim is to stop viral replication in infected cells and to stop viral spread to other uninfected cells. RNA interference represents the most promising approach in the treatment of infectious disease as it is one of the most effective means of preventing the proper functioning of target genes. The ultimate aim of anti-viral or anti-microbial therapy is to prevent the replication of the pathogen in question, and this is best achieved by targeting the molecular mechanisms that drive its replication. One can use RNA interference to specifically turn-off the expression of a pathogen's genes without affecting the gene expression of the infected cell. The result is that the pathogen is crippled and no longer able to spread to new cells. Effective RNA

interference can be achieved by either chemical means (where short interfering oligonucleotides are administered to infected cells) or genetic means (where vectors encoding for short hairpin oligonucleotides are administered). In both mechanisms the short oligonucleotides interact with the messages derived from the pathogen's genome resulting in their degradation and the inability of the pathogen to effectively express its genetic material. RNA interference in the treatment of infectious disease is at an early stage, but it represents the most promising novel therapy currently employed in the laboratory.