Term
| what has been the long term success with islet cell transplantation? |
|
Definition
the early results from a group of seven patients with T1DM were encouraging: after 1 year, 80% of the patients treated according to the Edmonton protocol no longer required exogenous insulin
unfortunately, however, a follow-up study from the Edmonton group, which included 65 patients with T1DM who underwent islet transplantation, found that by 5 years most of the patients had reverted to insulin dependence |
|
|
Term
| in what clinical situation does pancreas transplant usually occur? how successful is it? |
|
Definition
in most cases, either half or a whole vascularized pancreas is transplanted into a patient with end stage diabetic kidney disease, who also requires a simultaneous kidney transplant
with on going developments in the efficacy and safety of immunosuppressive regimens, complications realted to leakage of pancreatic juice have become infrequent after surgery
the long term outcomes of pancreas transplantation are approximately equivalent to those reported for kidney transplants |
|
|
Term
| where do the islet cells come from? |
|
Definition
isolate pancreatic islets are transplanted by direct injection into the portal vein, from whence they engraft in the liver and function as a source of insulin producing cells
removal of intact islets from cadaveric tissue is an extremely skilled and expensive job
even the best centers consider an extraction a success if they recover just 1/3 of the total islets; the remaining 2/3 either get missed or damaged during the extraction process
as a result, one donor organ is seldom sufficient to treat one recipient
the Edmonton protocol published in 2000 differed from previous islet transplantation protocols in 2 important ways
first, the Edmonton researchers selected patients who had not developed secondary diabetic complications
second, they used immunosuppression regimens designed to be relatively non-toxic to the transplanted islets |
|
|
Term
| what are the 3 main issues facing the use of embryonic stem cells to make new islet cells? |
|
Definition
the danger exists that un-differentiated embryonic stem cells inadvertently transplanted therapeutically (or derived by dedifferentiation of "unstable" differentiated cells) could become malignant and form teratomas, tumors composed of multiple cell types derived from one or more of the 3 germ layers
another important concern with the use of embryonic stem cells is that even if the parental stem cell was only weakly antigenic, the differentiated progeny might express major histocompatibility complex epitopes and become targets for allograft rejection
finally, the destruction of human embryos to facilitate production of embryonic stem cells or immortalized cell lines for research has been a major concern for many individuals on either ethical or religious grounds
such opposition has severely hampered the use of human embryonic stem cells in clinical therapies |
|
|
Term
| what is transdifferentiation, and how is it made to happen? |
|
Definition
transdifferentiation describes the conversion of a fully developed, mature cell of one type into a cell with a completely different phenotype
traditionally, only cells arising from adjacent regions in the developing embryo were though to have the potential to interconvert by transdifferentiation
for example, liver cells, which arise from the endoderm, can transdifferentiate into pancreatic beta cells, another cell type of endodermal origin
in 2006 a study demonstrated that adult fibroblasts could be reprogrammed to a pluripotent state, with many of the characteristics of embryonic stem cells
generation of these "induced pluripotent stem cells" (iPS cells) required the ectopic expression of 4 transcription factors using viral based vectors to transduce the mouse fibroblasts in vitro
dedifferentiate the cell first by forcing the expression of transcription factors away from the differentiated state and back to the pluripotent state |
|
|
Term
| what are the dangers with viral vector induced pluripotent stem cells? |
|
Definition
the disadvantages of using iPS cells are potentially the same as those for human embryonic stem cells but without the ethical concerns
the common method of delivering transcription factors by retroviral transduction also bears the risk of insertional mutagenesis and may render the transduced cells tumorigenic
however, now that iPS cells can be generated without the use of viral vectors or introduction of the cMyc oncogene, the main concern would be achieving satisfactory and appropriate differentiation and avoiding teratoma formation that could result from the presence of undifferentiated iPS cells |
|
|
Term
| how can gene therapy be used to cure diabetes? |
|
Definition
in gene therapy, the required gene (or genes) are introduced into cells ex vivo and the transduced cells transplanted into the recipient
alternatively, genes can be directly introduced in vivo into the target organ
use their tissue, differentiate out specific cells, study these cells. |
|
|
Term
| what does ectopic expression of the human insulin gene mean? |
|
Definition
infusion of the lentiviral vector encoding a human modified insulin gene into the portal system of liver rendered surgically ischemic resulted in long term cure of streptozocin induced diabetic rats
an unexpected finding was that ectopic expression of the human insulin gene in this system induced expression of transcription factors characteristic of pancreatic islet cells, which in turn enabled the liver cells not only to produce human insulin, but also to store and release it in response to changes in serum glucose concentrations |
|
|
Term
| what are the 2 genetic medicine approaches that show most promise for diabets? |
|
Definition
the first approach is the use of insulin and beta cell specific transcription factor genes, introduced into liver cells ex vivo and/or in vivo into the portal system, to transdifferentiate liver cells into cells with a beta-like phenotype
the second approach involves in vitro culture of autologous bone marrow to produce insulin secreting cell clusters that can be transplanted into the diabetic recipient
in order to obtain sufficient insulin producing cells to treat patients with diabetes mellitus, a combination of these 2 approaches may be worth investigating |
|
|
Term
| what is a major concern in transgenic insulin therapy, even if the insulin gene is correctly expressed? |
|
Definition
would adequate control of insulin storage and release be maintained?
the problem with injecting insulin in a diabetic is the timing with carbs
the glucose sensing system has to be maintained |
|
|
Term
| what has been the problem with using stem cells therapeutically? |
|
Definition
the technology posed moral questions b/c human embryos had to be destroyed to harvest the embryonic stem cells
the second problem was scientific
there was a mad rush to test the idea that specialized cells derived from stem cells could simply be transplanted into sick people (or animals) as cellular therapies to cure a host of diseases
"the big dream was to derive motor neurons from stem cells and then you would put them in the brain or spinal cord and the patients would just get up and start dancing"
but it did not work out that way in repeated animal experiments
"from beginning to end these experiments were failures" |
|
|
Term
| to model sickle cell anemia, what sort of pluripotential stem cell would you need? |
|
Definition
take a sickle cell patient and differentiate into a bone marrow type stem cell that is pluripotent (commited to a blood precursor) and take that up the erythroid lineage to study the production of hemoglobin
ALS:
extract skin tissue from adults, isolate specialized cells called fibroblasts from the sample, and then gently coax them with genes and chemicals to become nerve cells
1. skin cells are taken from a patient with ALS
2. researchers insert regulator genes into fibroblast cells from the skin's connective tissue
3. the foreign genes reprogram the fibroblasts into induced pluripotent stem cells, which are able to become many kinds of cells
4. iPOS cells clump together to form embryoid bodies. researchers add 2 small signaling molecules - retinoic acid and a substance that boosts cell division
5. the signaling molecules direct development of the embryoid body into a motor neuron/RBC
6. b/c the newly created nerve cells/RBCs were derived from a patient with ALS, they exhibit characteristics of the disease
7. trays of cells are prepared for robotic drug screening. one chemical compound is tested per well, allowing for broad sweeps of a variety of drug candidates |
|
|
Term
| what is the goal of the induced pluripotential stem cell procedure? |
|
Definition
| take an adult cell and biochemically coax it back into an embryolike state, allow it to replicate, and harvest stem cells from the resulting colony |
|
|
Term
| what advantage does the disease in a dish approach have over testing drugs in patients? |
|
Definition
many of these cell lines capture unique mutations found in people with unusually severe cases of LS
the disease in a dish approach is beginning to deliver on its potential, providing insights into the nature of motor neuron disease
in 2010 a preliminary screen of about 2000 compounds in ALS motor neurons from humans looking to see if any of the molecules would prolong the survival of nerve cells that contain the mutated ALS genes |
|
|
Term
| what is the difference between a lineage-committed cell and a terminally differentiated cell? |
|
Definition
lineage committed cell: limit their potential to specific tissue families; multipotent
terminally differentiated cells: mature body cells; locked into their identities |
|
|
Term
| in making iPSC with the latest techniques, does foreign DNA have to be integrated into the genome of the iPS cells? |
|
Definition
no
ultimately, researchers hope to produce iPSCs without using anytype of virus, but instead by simply exposing adult cells to a combination of drugs that mimic the effect of the reprogramming genes
chemicals can substitute the reprogramming genes in that each chemical activates a pathway of molecular interactions inside a cell that would be activated by the gene |
|
|
Term
| what is the difference between loss of function and gain of function single gene disorders? what's an example of each? |
|
Definition
hereditary gene disorders involve one or 2 mechanisms: loss or gain of function
in loss of function disorders, functionsl deficiency of a mutated gene leads to a protein deficiency, as in hemophilia and cystic fibrosis. in this case the therapeutic strategy consists of inserting a copy of the healthy gene.
in gain of function disorders such as Huntington's chorea and sickle cell disease, symptoms are linked to the synthesis, under the control of a mutated gene, of an abnormal protein that has deleterious effects. in this case, simply adding a healthy gene is not sufficient: the function of the mutated gene or its protein product must be inhibited. the best approach currently consists of repairing the implicated mutations in vivo |
|
|
Term
| in addition to the suicide gene approach, what transgenic approaches are used in cancer therapy to activate the immune system? |
|
Definition
on strategy consists of inserting a "suicide gene" into cancer cells that is capable of transforming a prodrug into a cytotoxic drug that kills the cancer cells
another technique is to insert a gene that produces a protein enhancing the uptake by cancer cells of a toxic substance administered via the bloodstream
alternatively, it is possible to insert a gene that enables the immune system to recognize and kill cancer cells
finally, the therapeutic gene can lead to the production of an angiogenesis inhibitor that prevents the formation of new blood vessel necessary to irrigate the tumor
by delivering a transgene gives you expression immune stimulating cytokines or delivering an antigenic target = suicide gene approach
has to be targeted at the tumor; the tumor cell is changing its environment to make it look more antigenic |
|
|
Term
| how could restoring p53 gene function in cancer cells work to treat the cancer? |
|
Definition
normal cells contain a gene designated p53, known as the "guardian of the genome"
this gene contributes to repairing mutations or, when the mutations are irreparable, induces the cell to self destruct
cells can become malignant when a mutation renders their copy of p53 non functional
in this situation the objective to to transfer a functional copy of p53 into cancer cells |
|
|
Term
| what genetic medicine approach is being used for cardiovascular disease? |
|
Definition
one of the therapeutic strategies envisaged for cardiovascular disease is to stimulate blood vessel formation by transferring genes encoding vascular endothelial growth factor (VEGF)
more vascularization in the diseased heart tissue |
|
|
Term
| why is it important that foreign genes do not get inserted in sperm or egg cells? |
|
Definition
to date, research aimed at human gene therapy has avoided manipulations that would deliberately affect descendants of the treated individuals, perhaps in unintended ways
the need for enlightened public debate over the merits and risks of germline therapy has however been made more urgent by the recent cloning of an adult sheep |
|
|
Term
| how are some anticancer gene therapy approaches similar to anticancer therapeutic antibody approaches |
|
Definition
| many proposed anticancer gene therapies under study take this last tack: they aim to induce cancer cells to make substances that will kill those cells directly, elicit a potent attack by the immune system, or eliminate the blood supply that tumors require for growth |
|
|
Term
| can gene therapy be used to block protein production, or only to replace missing proteins |
|
Definition
some gene therapygroups are also devising strategies to compensate for genetic mutations that result in destructive proteins
in one approach, called antisense therapy, short stretches of synthetic DNA acts on messenger RNA transcripts of mutant genes, preventing the transcripts from being translated into abnormal proteins
related tactics deploy small RNA molecules called ribozymes to degrade messenger RNA copied from aberrant genes |
|
|
Term
| what essential property must "smart" gene therapy vectors have? |
|
Definition
delivery of genes in human subjects is sometimes accomplished directly by putting vectors (agents carrying potentially therapeutic genes) straight into some target tissue in the body (in vivo)
more often the ex vivo approach is use: physicians remove cells from a patient, add a desired gene in the laboratory and return the genetically corrected cells to the patient
an in vivo approach still in development would rely on "smart" vectors that could be injected into the bloodstream or elsewhere and would home to specific cell types anywhere in the body
target cell specificity - key to limit gene expression where it should be |
|
|
Term
| what is the main advantage of ex vivo gene therapy? |
|
Definition
scientists remove cells from a selected tissue in a patient, expose them to gene transfer vectors in the laboratory (ex vivo) and then return the genetically corrected cells to the individual
can have a cell population that is as pure as you want = greater targeting capability
for tissues that are readily available this is the preferred way to do gene therapy
other tissues you don't have the option of ex vivo therapy
can fairly easily purify the cells you want (T cells, dendritic cells, stem cells) and made into homogenous samples |
|
|
Term
| can the expression of genes introduced by gene therapy be precisely regulated |
|
Definition
| biologists have yet to achieve such precise control over foreign genes put into the body |
|
|
Term
| why are viruses particularly well suited to serve as gene therapy vectors? |
|
Definition
virsus - which are little more than self replicating genes wrapped in protein coats - have drawn the most attentions as potential vectors
they are attractive b/c evolution has designed them specifically to enter cells and express their genes there
viruses are the simplest gene replication machines there are
they have evolved to be able to enter cells, deposit their genome, take over the metabolic system of the cell to make more viruses |
|
|
Term
| what sorts of genetic engineering is done to make viral vectors? |
|
Definition
scientists can substitute one or more potentially therapeutic genes for genes involved in viral replication and virulence
in theory, then, an altered, tamed virus should transfer helpful genes to cells but should not multiply or produce disease |
|
|
Term
| what are the main differences between a natural viral life cycle and the ideal life cycle for a viral vector of gene therapy? |
|
Definition
basic life cycle of a naturally occurring virus:
naturally viruses infect, replicate, burst the cell
virus enters cell -> inserts viral DNA into the host DNA -> viral proteins and viral genes are made -> assembly of new viral particles leave the host cell -> spread of infection
life cycle of ideal gene therapy vector:
you do get infection, and deposition of the genome with the transgene, but you only get expression of the transgene product (no viral components), no possibility of viral replication
vector enters cell -> inserts DNA into host's DNA -> therapeutic protein is made with no unwanted viral proteins and no viral genes -> no new viruses are made |
|
|
Term
| what are the main advantages and disadvantages of retroviral vectors |
|
Definition
advantages: integrate genes into host chromosomes, offering chance for long term stability
disadvantages: genes integrate randomly, so might disrupt host genes; many infect only dividing cells; the physical act of inserting a transgene can disrupt the normal gene; in the process of integrating the retrovirus can also turn on genes (has caused leukemia in clinical trials) |
|
|
Term
| what are the main problems with liposomal vectors or naked DNA? what is a particular problem for naked DNA? |
|
Definition
liposomes: less efficient than viruses at transferring genes to cells
naked DNA: inefficient at gene transfer, unstable in most tissues of the body
in efficient so have to give a lot to have an effect |
|
|
Term
| where in the chromosome do retroviruses insert their DNA |
|
Definition
| retroviruses splice their DNA into host chromosomes randomly, instead of into predictable sites |
|
|
Term
| what cellular event must occur for retroviral DNA integration to occur, and when does that happen? |
|
Definition
current retrovirus vectors reach chromosomes only when the membrane surrounding the nucleus of the host cell dissolves, an event that occurs solely during cell division
the exception is HIV: can infect and deliver its DNA and deliver it into the chromosome without the cell dividing |
|
|
Term
| what parts of the virus have to be genetically engineered to target the gene therapy to specific cells? |
|
Definition
| to make retroviruses more selective about the cells they invade, investigators are learning how to replace or modify natural envelope proteins or to add new proteins or parts of proteins to existing envelopes |
|
|
Term
| why is the HIV retrovirus considered a candidate for a gene therapy vector? |
|
Definition
| HIV, a retrovirus, has the ability to deposit its genes into the nucleus of nondividing brain cells without waiting for the nuclear wrapping to dissolve during cell division |
|
|
Term
| which viral vector is most versatile and efficient? |
|
Definition
human adenoviruses have gained the most popularity as alternative to retroviruses in part b/c they are quite safe
the naturally occurring forms typically cause nothing more serious than chest colds in otherwise healthy people
moreover, they infect human cells readily and , initially at least, tend to yield high levels of therapeutic protein |
|
|
Term
| for what therapies would transient gene expression make sense? |
|
Definition
| when a protein is needed only temporarily to induce an immune response to cancer or to a pathogen - short term expression of a foreign gene may be preferable |
|
|
Term
| what would you expect to see in clinical trails if patients rapidly become immune to viral vectors? can anything be done to deal with that issue? |
|
Definition
during an initial round of treatment, such vectors might infect the appropriate cells and generate high amounts of the desired proteins
but soon host defenses come into play, killing the altered cells and inactivating their new genes
further, once the immune system is alerted to the virus, it eliminates them quickly when they are delivered a second time
such responses probably have contributed to a shut down of gene expression in a number of adenovirus gene transfer studies in patients |
|
|
Term
| which gene therapy approach would be best protein replacement in hemophilia, for example? |
|
Definition
if a patient inheritied a genetic defect and needs a continuing supply of the normal gene product throughout life, a vector that can integrate the therapeutic gene into the patient's chromosomes, where it will stay in perpetuity, might be test
then a retrovirus or adeno-associated virus may be selected; want stable permanent expression |
|
|
Term
| what can happen to a protein, produced by gene therapy, in an adult who has never been able to make a protein naturally |
|
Definition
we still have few clues as to how the defensive systems of patients will respond when they encounter a seemingly foreign protein from a therapeutic gene
to prevent an inactivating immune reaction, physicians might have to treat some patients with antirejection drugs to try to induce immune tolerance to the encoded protein by carrying out gene therapy very early in a patient's life (before the immune system is fully competent) |
|
|
Term
| in engineering oncolytic viruses, what is the trade off that has to be optimized? what are some workarounds for suboptimal viruses? |
|
Definition
nearly everyone has been exposed at one time or another to adenoviruses, so almost all of use carry antibodies the immune systme makes to target them for destruction
accordingly, shots of adenoviruses as cancer therapies might cause severe, flulike symptoms if the body recognizes them as foreign and ramps up an immune response to eradicate them (wiping out the viruses would also squelch the therapy)
at the same time, recognition by the immune system ensures that the viruses to not reproduce out of control
infestigators are now designing various therapeutic approaches to optimize the efficacy of virotherapy and minimize the chances that adenoviruses will cause side effects
these strategies include giving immunosuppressive drugs at the time of virotherapy and modifying the adenoviruses so that they do not trigger a reaction by the immune system
for oncolytic therapy you want immune recognition b/c you want to have an equilibrium b/c if any normal tissue is being attacked, you want the immune response to attack that, but not strong enough that it destroys the therapy |
|
|
Term
| what is the difference between transductional and transcriptional targeting? |
|
Definition
transductional targeting - researchers are attempting to adapt the viruses so that they preferentially infect, or transduce, cancer cells
they are attaching adapter molecules onto the viral outer coal proteins or directly modifying these proteins to try to prevent the viruses from entering normal cells and instead prompt them to home in on tumor cells
transcriptional targeting - involves altering the viruses so that their genes can be active, or transcribed, only in tumors
the engineered viruses can enter normal cells, but they cannot reproduce and kill them
once they enter cancer cells, however, the tumor specific promoter lets them make millions of copies of themselves and ultimately burst the cancer cells where they can spread to and destroy other tumors |
|
|
Term
| how can adenoviruses be altered so that normal cells do not get infected? |
|
Definition
transductional targeting is particularly necessary b/c, unfortunately, adenoviruses bind more efficiently to the variety of normal tissues in the human body than they do to most tumor cells
we can reverse this pattern using specially generated adapter molecules made of antibodies that snap onto the arms of the virus like sockets on the socket wrench
by attaching carefully chosen antibodies or other molecules that selectively bind to only a specific protein found on tumor cells, we can render adenoviruses unable to infect any cells but cancerous ones
alter their ligands for the target cell; you can re-engineer the ligands so they don't recognize the normal cells, block the ligands |
|
|
Term
| what transmembrane protein could serve as a receptor for oncolytic viruses? what is a drawback with this approach? |
|
Definition
adenoviruses can bind to cellular proteins called integrins
these molecules help cells stick to the network of connective tissue, called the extracellular matrix, that organizes the cells into cohesive tissues
although integrins are also made by healthy cells, cancer cells produce then in abundance as they become metastatic and begin to squeeze through tissue layers and travel throughout the body
drawback to integrins: lots of cells have integrins (especially immune cells/leukocytes), lack of specificity |
|
|
Term
| which tissue specific enzyme can be used for promoter targeting of melanoma? what side effect might this approach have? |
|
Definition
the promoter for the key enzyme making melanin gets turned on in melanocytes but generally is off in most other body tissues
in the dealy skin cancer melanoma, the gene encoding this enzyme is fully functional, making the tumors appear black
adenoviruses have been engineered with a promoter for the enzyme adjacent to genes that are essential for the virus's ability to replicate
although these viruses might infect normal cells, such as liver cells, they can reproduce only inside melanocytes, which contain the special combination of proteins needed to turn on the promoter
adenoviruses engineered to contain the promoter for the enzyme that makes melanin can also replicate in normal mealnocytes, so on their own they might cause spots of depigmentation
and adenoviruses that are designed to bind to receptors on the surfaces of tumor cells can still invade a small proportion of healthy cells |
|
|
Term
| what is a good promoter target that should be specific to certain liver cancers? why is this a better target than the melanin enzyme promoter in melanoma? |
|
Definition
in liver cancers the promoter for the gene alpha-fetoprotein - which is normally shut down after fetal development - becomes reactivated
adenoviruses containing that same promoter hold promise for eradicating liver tumors |
|
|
Term
| what would be a good transcriptional target for prostate cancer? what would be the drawback? |
|
Definition
researchers used adenoviruses that had been engineered to contain the promoter for prostate specific antigen, a protein made in abundance by prostate tumors
they administered the virotherapy to 20 menb who received varying doses of the adenoviruses
none of the men experienced serious side effects and the tumors of the 5 men who received the highest dose of the virotherapy shrack by at least 50%
PSA is not exclusively in prostate tumor cells, made by normal cells as well |
|
|
Term
| which of therapy is transductional targeting? |
|
Definition
| carries the gene for granulocyte macrophage colony stimulating factor, an immune system stimulant |
|
|
Term
| which therapy is transcriptional targeting? |
|
Definition
targeted to prostate cancer cells using prostate specific promoters
harbors 2 gene deletions that prevent it from reproducing in normal cells
has a gene deletion that restricts it to actively dividing cells such as cancers
able to replicate only in cancer cells bearing the activated oncogene ras |
|
|
Term
| how could you use viruses to make cancer cells vulnerable to chemotherapeutic prodrugs? what is the draw back of the approach described here? |
|
Definition
researchers are also arming therapeutic viruses with genes that make the cells they infect uniquely susceptible to chemotherapy
the technique involves splicing into the viruses genes that encode enzymes that turn nontoxic precursors or prodrugs into noxious chemotherapies
in one example an adenovirus carried genes encoding the enzymes capable of converting prodrugs into anticancer compounds camptothecin and 5FU
the scientists engineered the viruses so that they could make the enzymes only in actively dividing cells, such as cancer cells
drawback: may not be 100% specific for cancer cells; collateral damage |
|
|
Term
| what protein must be expressed only in tumor cells to take advantage of the tumor specific gene promoter? |
|
Definition
the cancer cells are expressing a transcription factor that is not expressed in normal cells
since the normal cells don't have this transcription factor, the promoter cannot be activated |
|
|
