Cell and Gene Therapy

What is the aforementioned process called?

*Induced pluripotency*

Cytotoxic chemotherapy is more effective against which type of cancer cell?

*Tumor cells* rather than cancer stem cells

What makes the concept of using mesenchymal stem cell therapy so appealing?

Ability of MSCs to differentiate into a number of important tissue types (cardiac, nerve, muscle, bone) Relative ease with which MSCs can be isolated from peipheral blood or adipose tissue

Bone marrow is relatively ______ in stem cells, which reduces the need for significant ______ in culture and marrow can harvested from donors and delivered patients with reasonable target tissue ________

Abundant Expansion Selectivity

Another way of avoiding ethical problems associated with therapeutic use of ESCs is to make use of stem cell populations present in _________ (______ stem cells) for therapeutic purposes

Adults Somatic

These viral vectors must elicit a minimal...

Allergic/inflammatory response

Bone marrow donation can be either *autologous* or ________

Allogenic

What is a benefit of ex vivo gene therapy?

Allows for a wider range of methods to introduce therapeutic genes, including standard DNA *transfection* Greater ability to characterize modified cells peior to reintroducing them to the patients

Adult stem cells divide (symmetrically/asymmetrically). What do the daughter cells look like?

Asymmetrically One daughter cell retains the properties of the parent stem cell Second daughter cell that is capable of differentiating into the cell type necessary to *replace depleted cells*

MSCs are co-resident with hematopoietic stem cells in bone marrow, and seem to ________ the immune response

Attenuate

Lymphomas are treated with _______ transplants and leukemias are treated with _________ transplants

Autologous Allogenic

Induced pluripotent stem cells bypass ____ formation, which alleviates many _______ concerns

Blastula Ethical

Embryos lose totipotency in the ________ stage, but the inner cell mass (epiblast) of the blastocyst retains the capability to generate all organismal, rendering it _______

Blastula Pluripotent

What is the exception to the challenges of using adult stem cells?

Bone marrow transplantation (BMT), which is the most widely performed stem cell therapy

Future prospects of in vivo and ex vivo therapies have been boosted by the discovery of _________, which allows highly specific excision and/or delivery of genetic material at any positions in the genome

CRISPR/Cas9

What does the stem cell theory of cancer state?

Cancers aries from dysregulated adult stem cells rather than through mutation of somatic cells

In ex vivo, cells with the therapy can be _________ extensively in order to determine the site of integration and minimize the chances that cells with malignant potential are transplanted into patients

Characterized

Modifying T cells with ______, single chain antibodies raised against antigens that are commonly expressed on utmor cells (*chimeric antigen receptor T cells) elicits a robust ________response that has increased options and outcomes

Chimeric Anti-tumor

Immunological activity of the graft can be beneficial in what way?

Clearance of surviving tissue that might otherwise contribute to patient relapse, called the *graft versus tumor* effect

The ability to generate embryos by SCNT that are genetically identical to the donor rasies additional concerns about human _______

Cloning

Macrocyclic immunosuppressive agents such as *ciclosporin A* and *Tacrolimus* form complexes with endogenous target proteins _____ and ______ respectively, which bind to and inhibit ______

CpN FKBP Calcineurin

Total myeloablative conditioning regimens involve cytotoxic agents such as _______ and _______ in combination with total body irradiation (TBI)

Cyclophosphamide Fludarabine

What two indicents slowed in vivo gene therapy for a while?

Death of a patient from an acute reaction Leukemia development

What is *in vivo gene therapy?*

Delivery of a therapeutic gene to the patient

What is *ex vivo gene therapy?*

Delivery of therapeutic gene to explanted cells, which are returned to the patient following introduction of the therapeutic gene

What is key in *in vivo gene therapy?*

Design of safe, effective recombinant viral vectors for delivery of therapeutic genes to patients

What are the three methods by which donor tissue can be extracted for bone marrow transplantation?

Directly from a donor *Hematopoietic and mesenchymal stem cells* can be harvested from peripheral blood through *apheresis* Umbilical cord blood can be used as a source of hematopoietic and mesenchymal stem cells

The resulting autologous transplant from iPS (does/does not) require immunosuppression

Does not

Consenting patients could eventually donate for research purposes to foster the creation of _________ stem cell lines

Embryonic

Although SCNT reduces the need for immunosuppression following autologous transplantation, the method still involves generating stem cell line from ______, which carries a similar ______ concern

Embryos Ethical

Pluripotent stem cells for research derive from which three sources?

Epiblast cells donated by consenting in vitro fertilization patients (*embryonic stem cells*) Embryonic stem cells generated through *somatic cell nuclear transfer (therapeutic cloning)* Pluripotent stem cells produced through genetic or small molecule reprogramming of somatic cells (*induced pluripotent stem cells*)

Since stem cells from the hematopoietic system can be collected relatively easily, diseases of this system have been targeted extensively for ________ gene therapy

Ex vivo

How can asymmetry be imposed?

Extrinsically through interactions with a *stem cell niche* Intrinsic regulation

What are the challenges to working with adult stem cells?

Few in nummber Less robust self renewal properties than pluripotent

Viral delivery systems should integrade therapeutic ______ into specific sites of the patient's genome to avoid introducing alterations that could give rise to ______ consequences

Genes Deleterious

Chemical approaches to iPS have also been developed, but the _____ approaches predominate

Genetic

In addition to its use to mobilize hematopoietic and mesenchymal stem cells prior to bone marrow donation, recombinant forms of GM-CSF such as *pegfilgrastim* are given to do what?

Given to immunocompromised patients to help protect against infection

What is the graft rejection called when the graft attacks the host?

Graft vs. host disease

The efficiency of harvesting stem cells from peipheral blood is enhanced when donors are treated with what prior to apheresis?

Granulocyte-macrophage colony stimulating factor (GM-CSF)

Mesenchymal stem cell therapy (Prochymal) offers an alternative approach for treating _______

GvHD

What issues to these challenges pose to working with adult stem cells?

Harvesting them from donors is more difficult Makes expanding them in culture to achieve critical mass more *time consuming* Increases the risk of contamination or *oncogenic mutation*

What caused the delay for directed differentation research to begin?

Human ESCs required extensive optimization Ethical issues

How is graft vs. host disease usually treated?

Immunosuppression with macrocyclic compounds that *interfere with T-cell activation* and IL-2 production

Recently, ex vivo therapies have been explored to improve cancer _________

Immunotherapy

Notable failures of using MSCs have resulted in: What does this highlight?

Kidney cancer Blindness The challenge to translating adult stem cell therapy to the clinic and the unique features of bone marrow and hematopoietic stem cells that contribute to the widespread success of bone marrow transplantation

Stem cells are characterized by ______ capacity for *self-renewal* and relative resistance to cell ______ pathways

Limitless Death

Upon gastrulation and formation of three germ layers, the pluripotency is ________, and resident stem cells are limited to production of the cell types derived from...

Lost That particular germ layer

Ex vivo, when applicable, is beneficial because it eliminates...

Most of the challenges seen in in vivo gene therapy

The use of nuclei from somatic cells carries the possibility that a given clone might carry spontaneous _________ in its genome that could be detrimental to the long term ______ of the transplanted stem cells or the recipient

Mutations Viability

Recipients of bone marrow transplants are typically treated with radiation and chemotherapy called ___________ conditioning, in an effort to eliminate cells contributing to whatever lymphatic disorder prompted BMT, and to provide a site for transplant _________

Myeloablative Engraftment

Because of these effects, care teams much optimize which two treatments?

Myeloablative conditioning Immunosuppression

GM-CSF is the primary growth factor controlling expansion of _______ precursors through activation of which pathway?

Myeloid Jak/STAT

Inhibition of calcineurin leads to prevention of _______ dephosphorylation, nuclear translocation and transcriptonal activation

NFAT

T-cell receptor engagement promotes...

Opening of calcium channels --> Activation of the phosphatase calcineurin --> Removal of inactivating phosphorylation Nuclear translocation of nuclear factor in activated T cells (NFAT) Transcription of cytokines such as IL-2 that mediate T-cell dependent immunological responses

The failure to differentiate ALL transplanted stem cells can lead to what?

Outgrowth of pluripotent cells that are resistant to programmed cell death

An alternative method for generating ESCs was published, enabling the creation of _______-derived stem cells (*autologous*) which has the potential to obviate the need for _________

Patient Immunosuppression

Somatic cells used for iPS are typically derived from...

Patients

Some research, though not definitive, suggests that adult stem cells can generate cell types outside of their tissue lineage of origin, which is referred to as ______

Plasticity

Since pluripotent stem cells resemble cancer stem cells, transplantation of undifferentiated, _______ stem cells carries the risk that patients may develop transplant-derived cancers

Pluripotent

In vivo gene therapy is usually carried out using ________ viruses as delivery systems

Recombinant

Patients who undergo allogenic BMT also must be monitored for signs of transplant _______, although in this case the transplant supplies the _________ reaction

Rejection Immune

Intense conditioning can reduce the risk for _______, but may be difficult for some people to tolerate, such as the elderly or those with comorbidities

Relapse

What did this newer method involve?

Removing the haploid nucleus from an unfertilized egg and replacing it with a patient-derived somatic cell (*somatic cell nuclear transfer SCNT*)

What do resident stem cells do?

Replace cells lost to injury or normal clearance

What is the function of adult stem cells?

Replace cells that are lost through normal cell turnover and shedding in order to maintain tissue homeostasis

The delivery of a small number of genes to human somatic cells by transfection or viral transduction is sufficient to do what?

Reprogram their phenotype from *differentiated to stem cell*

What does this biased effectiveness lead to?

Response and relapse seen with this therapy

Immunosuppressive agents carry significant liabilities, such as what? This makes it very important to carefully manage the medications.

Risk of infection and cancer Cardiac and nephrotoxicity

A fertilized ovum functions as a ______ from which *all other cells can grow*. It must have the ______ to develop into every cell type observed at every stage of life

Stem Potency

Further differentiation and *hierarchical fate restriction* produces somatic or *adult _________ _____* that are capable of producing numerous cell types, thus making them _______

Stem cell Multipotent

The concern of malignancy is rooted in which cancer theory?

Stem cell theory of cancer

Rapamycin (sirolimus) also forms a complex with FKBP family members, but...

That complex binds to mTOR and mediates its immunosuppressive effects through *cell cycle inhibition* via CDK2

An alternative approach for patients with germline mutations that lead to loss of normal function or gain of aberrant function is to replace...

The mutant gene with a wild-type copy

What is the major limitation of all therapeutic approaches based on transplantation of pluripotent stem cells?

The risk of malignancy

The ability to rapidly generate patient-derived iPS creates opportunities to predict patient response to therapy, screen for novel ___________ modalities or model normal development or ________ abnormalities

Therapeutic Congenital

What else is this method referred t as?

Therapeutic cloning

What is an additional limitation of embryonic stem cells in therapeutic applications?

Tissue is always derived from a donor that is genetically and immunologically different from the recipient (*allogenic*) thus requiring *immunosuppression* to prevent graft rejection by patients

Cells that are capable of generating every possible cell in the organism are referred to as _______, and this potency is retained through the ______ stage

Totipotent Morula

(True/false): CAR T-cells are rapidly emerging as a pardigm for cancer immunotherapy

True

(True/false): The advent of reduced intensity and non-myeloablative conditioning has made BMT accessible to more patients

True

Tissues counterbalance ______ (skin, gut, blood) and maintain ________ capacity (brain, liver, muscle, vasculature) through the existence of ______ stem cells

Turnover Regenerative Resident

Since these indicents, major developments have been made in ______ design, and *Glybera* has already been approved in Europe

Vector

In ex vivo, immune responses to _______ vectors that compromise gene delivery are avoided as are potentially ________/inflammatory responses in patients

Viral Allergic

Hundreds of embryonic stem cell lines were rapidly generated and conditions were established enabling *_______ differentiation* of embryonic stem cells in vitro into numerous cell types

*Directed*