Gene therapy and immunology Gene Therapy – an approach designed to treat disease by replacing, altering or supplementing genes that are defective or missing. Two immunological factors play important roles in gene therapy: Immunodeficiency diseases are prime candidates for therapeutic approaches where genes are introduced into a patient to correct a gene mutation. Immune responses against vectors used in gene therapy can be important for the success or failure of a particular approach.

Correcting an immunodeficiency disease: X-linked SCID A clinical study in France (1999) sought to cure young patients with X- linked SCID. Approach: isolate stem cells from bone marrow, grow in culture, replace defective  c gene, and reintroduce into patient. Isolate CD34+ cells add  c gene Patient cells with receptors restored

Ex vivo therapy - cells with defective genes are removed from a patient for transfection (introduction of genes). The treated cells are then returned to the patient. In vivo therapy – direct administration of a gene or packaged gene to the patient. Vector - a delivery device for insertion of a gene into cells. Viral vectors are currently the most common means for efficient gene transfer.

Retrovirus vector Retroviruses have a small RNA genome that has three genes coding for structural proteins (Gag, Pol and Env) required for viral replication surrounded by two long terminal repeats LTR  Gag Pol Env LTR LTRs contain promoter and enhancer functions for viral transcription and are involved in integration of the virus into the host cell genome. A short packaging sequence psi (  ) is needed for the RNA transcript to be packaged into a mature virion.

Viruses engineered for therapy lack genes needed for replication, these are replaced by the therapeutic gene, but retain LTR’s (needed for expression) and the packaging sequence. The essential functions of gag, pol and env are provided by stable packaging cell lines that express these proteins and allow the defective virus to be packaged into a mature virion.  cDNA of interest  neo resistance cDNA promoter Gene of interest can be driven by a viral promoter or by a different inserted promoter. A selected promoter might be advantageous if someone wanted tissue-specific expression of a particular gene. Can also include antibiotic selection marker to select infected cells for ex vivo experiments.

Retroviral vectors have a couple of limitations: 1). they only infect dividing cells 2). they integrate more or less randomly into the host genome. The right handed LTR can promote and enhance expression of genes adjacent to it. If one of these is a proto-oncogene, could potentially activate it inappropriately. Risk of causing cancer? cellular DNA proviral DNA cellular DNA (or transduced gene)

In the French study, retroviral gene transduction led to successful restoration of functioning immune systems to 9 of 11 boys with X-SCID. Boys developed normal numbers of CD4 and CD8 T cells and responded normally to childhood immunizations. However, in September of 2002, one of the 11 children developed leukemia three years after initiation of the project. The leukemia arose from expansion of a single  T cell and was due to insertion of the viral vector near a gene known as LMO2, which is a transcription factor linked previously to leukemia. It was unclear as to whether or not this was a highly unusual event or might be more common. Some trials were temporarily suspended. Then, in December of 2003, a second one of the 11 children developed leukemia due to expansion of an  T cell, also due to viral vector insertion near LMO2. Unfortunately this year a third child from the original French study also developed leukemia, but the mechanism appears to be different and not involve LMO2. This has caused temporary suspensions of related trials on a world-wide basis.

Adenosine deaminase deficiency ADA-SCID is a recessive disease that leads to an accumulation of toxic purine metabolites that affects multiple cell types, but particularly lymphocytes. This was the very first genetic disease to be treated with a gene therapy approach in Patients with ADA-SCID are best treated with a bone marrow transplant (not always possible to find good match). An alternative treatment is to give the patients ADA-PEG, adenosine deaminase coupled to polyethylene glycol to help stabilize the enzyme. This is not curative, but does ease symptoms.

The original gene therapy approach was similar to that discussed above for X-linked SCID except that T cells were isolated and these were infected with the retrovirus containing the cDNA for adenosine deaminase. patient add ADA gene Isolate T cells grow in culture in IL-2 patient cells with enzyme restored For ethical reasons, the patients were also given ADA-PEG as a back- up therapy. Later trials used CD34 + stem cells rather than T cells

The trials suffered from two problems: 1). The percentage of ADA expressing cells that showed up in the periphery was very small. 2). it was difficult to evaluate the effectiveness of the therapy due to the complicating factor of providing the ADA- PEG. Overall improvement in immune responses was observed. However, discontinuation of the ADA-PEG did cause conditions to worsen.

Two adjustments to increase efficacy of approach: 1. Patients no longer given ADA-PEG. The reasoning is that infected cells re-administered to the patient will have a growth advantage over ADA- deficient cells and will more readily populate the bone marrow. The provision of ADA-PEG eliminates much of this advantage 2. Patients treated with a low intensity, nonmyeloablative therapy to partially destroy their immune system to make room in the bone marrow for the infected cells to expand. Isolate CD34 + cells add ADA gene Patient with partial depletion of bone marrow cells cells with enzyme restored ADA+ cells have a growth advantage

Adenovirus vectors. Adenoviruses cause infections in humans that are relatively mild, often causing upper respiratory symptoms. However, as a consequence, they have the potential to trigger inflammatory responses. Adenovirus vectors are widely used in gene therapy because they exhibit the highest transfection efficiencies both ex vivo and in vivo. They can infect both dividing and nondividing cells of a wide variety of cell types. In in vitro cell culture, adenoviruses can replicate to very high titers so that sufficient quantities of vector can be prepared for clinical trials.

The genome of adenoviruses: Linear, double-stranded DNA. Viral DNA is encapsulated within a protein coat (non-enveloped virus). Adenovirus DNA does not integrate into host chromosomal DNA and remains an episome (separate DNA in nucleus) in cells. Thus, cannot cause insertional mutagenesis. The virus has two general sets of genes, early and late, which are expressed either before viral replication (early) or after (late) in a host cell. The first-generation viruses had one or more early genes (typically E1 or E3) replaced by the gene of interest. The E1 or E3 gene products could be supplied by packaging cells to make virus for therapy. E1a, E1b E3 ITR promoter cDNA (inverted terminal repeat)

Adenovirus vectors can trigger cytotoxic T cell responses directed at transfected cells expressing adenovirus proteins followed by a humoral response generating anti-adenoviral antibodies. These two characteristics of adenovirus vectors permit only transient expression of therapeutic genes and limit the ability to repeatedly administer virus.

This potential problem really came to light with a recent incident at the University of Pennsylvania. An 18 year old patient by the name of Jesse Gelsinger had volunteered to participate in a clinical gene therapy trial. Jesse suffered from an ornithine transcarbamylase deficiency (a urea cycle enzyme) that can lead to elevated blood ammonia concentrations. Was kept in check relatively well with a very low protein diet and drug treatment. Jesse was treated with an adenovirus vector carrying the ornithine transcarbamylase gene that was injected directly into an artery leading to his liver (these are the cells that should have OTC). He should have suffered only mild, flu-like symptoms, but somehow in this patient the adenovirus triggered an overwhelming systemic inflammatory response causing acute respiratory distress and death due to lung failure and anoxia. He survived only four days following the procedure. Follow-up studies showed that much of the virus failed to infect liver cells, but either infected liver macrophages or passed through the liver to lymph nodes, spleen and bone marrow.

More recent versions of adenovirus have been developed that are termed “gutless” vectors because they are missing most all viral genes, but still contain inverted terminal repeats and a packaging sequence around the transgene. All other required genes needed for production of the virus for use in therapy can be supplied by co-infecting helper viruses. These vectors present fewer proteins to the immune system and can prolong gene expression. Immune responses will likely still be problematic with time.

Adeno-associated viruses (AAV) as vectors. Small parvovirus often found in cells infected with adenovirus. Has only a single 4.7 kb single-stranded DNA genome surrounded by a protein coat. The DNA does integrate into the host genome, but does so at only a single site on chromosome 19. Virus can infect many cell types, both dividing and nondividing. The AAV proteins expressed are nontoxic to cells and do not trigger a host immune response, so they do not cause inflammation like adenoviruses. Can remove most of genetic information with exception of two 145 bp terminal repeats and replace with gene or genes of interest. (however, it can’t be any larger than the wild-type virus, so this limits the amount of DNA you can incorporate)

AAV can’t reproduce by itself, but needs help from other viruses like adenovirus or herpes virus (that’s why it is only seen in cells also infected by other viruses). This made it tough to figure out how to propagate the virus in culture. Investigators have identified the required helper genes and put these into packaging cells to allow production of helper-free AAV. TRRep Cap TR Wild-type virus has two genes: rep and cap. Rep contains information for replication, expression and integration. Cap has genes for capsid structural proteins. Surrounded by terminal repeats containing promoter. Replace with transgene and any regulatory sequences needed. TR promotercDNA

Can get stable expression in infected cells (but virus does not replicate and spread, would be temporary). Unfortunately, without Rep, the virus tends to integrate somewhat randomly, but seems to prefer transcribed genes. Could cause insertional mutagenesis.

Other viral vectors being examined: Herpes Simplex virus vectors are of interest because HSV has the capacity to infect neurons. Potentially useful for treatment of brain disorders like Parkinson’s disease and brain tumors like gliomas. Lentiviruses are retroviruses, but are much more complex in their genomes, containing 6 additional genes. HIV is a lentivirus. The packaging cell line is much more complex due to additional gene requirements. However, lentiviruses have the advantage that they can infect nondividing cells as well as dividing cells.

Nonviral vectors for gene therapy-viral vectors all elicit an immune response to some degree, have potential toxicity issues (e.g., insertional mutagenesis, inflammatory reactions), and are sometimes difficult to produce in large amounts. Nonviral delivery systems could potentially circumvent many of these problems. Naked DNA –Plasmid is injected or delivered via gene gun. Often suffers from a low transfection efficiency, especially compared to viruses -of considerable interest in the vaccine field. New studies suggest that simple iv injections can deliver to muscle and liver better than anticipated. Cationic Lipids and Liposomes - When plasmid DNA is mixed with small liposomes containing cationic lipids, lipid-plasmid DNA complexes form due to electrostatic interactions between negatively charged phosphate groups of DNA and positively charged lipid residues. These DNA-cationic lipids can enter cells through endocytosis. A drawback is low transfection efficiency in vivo. Most DNA transfected into cells with cationic lipids remains in endosomes. Can include targeting molecules in the liposomes to deliver them to specific cell types. Can add polyethylene glycol derivatives to phospholipids at the liposome surface to produce “stealth liposomes”, which circulate in the bloodstream for extended periods. Some investigators have tethered antibodies to the transferrin receptor to the polyethylene glycol covering to allow the liposomes to cross the blood-brain barrier.

RNA interference There is a process known as posttranscriptional gene silencing that was first identified by investigators trying to create a dark purple petunia. Adding genes for one color tended to turn off other color genes in an odd fashion – it did so after the genes were transcribed. The intermediate in the process turned out to be a double-stranded RNA molecule. The process has been found to be highly conserved in eukaryotes and probably persists in part due to its role in viral defense. It was subsequently shown in C. elegans (nematodes) that injection of a dsRNA corresponding to the mRNA coding for a particular protein could actually eliminate production of that protein. The dsRNA was processed to form small oligonucleotide fragments by an enzyme known as Dicer. These fragments bind to a complex known as the RNA-induced silencing complex or RISC. This complex targets the homologous mRNA and cleaves it.

It was found that this also works in human cells, with the complication that, if you give a human cell a dsRNA, you will induce the interferon response and shut off lots of protein synthesis. However, it was found that if you give the cell only small dsRNA oligonucleotides, the interferon response does not occur. Thus, it is possible now to eliminate the transcription of specific proteins form cells. A powerful tool for experimentalists. The process is called RNA interference or RNAi and the small oligos are called small, interfering RNAs or siRNA. It is also possible to express a small hairpin RNA that is processed by dicer to form the siRNAs. These are referred to as shRNAs or short, hairpin RNAs. Allows one to use plasmids as vectors or viruses as vectors for the delivery of these into cells. Consequently, there is considerable interest in using these as therapeutic agents for the treatment of diseases where the elimination of a particular protein is sought (e.g., cancer, HIV, Huntington’s disease).

Interferon response

Gene therapy trials. There are currently on the order of clinical gene trials underway, mostly in the U.S % of these target cancer, about 9.5% target monogenic diseases (disease caused by single gene mutation), 8% target vascular disease and 6.6% infectious diseases. 2. The most common gene delivery method being tested is retrovirus (27%) followed by adenovirus (26%) and naked DNA (15%). 3. The most common transgene being delivered is a cytokine gene (e.g., lymphocytes transfected to enhance tumor killing power and replaced) followed by antigens (enhance tumor immunity) and then genes for tumor suppressors. 4. The most common delivery route is intratumoral followed by iv. Most are in Phase I trials which test mostly for safety.