Neural Stem Cell-based Gene Therapy

Gene therapy comes of age:
edit genomes to cure diseases
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Retrieved from:Web: ExtremeTech

Malignant brain tumors can be grouped into two categories base on its cell origin. Primary brain tumors arise from the various cells that make up the brain and central nervous system while secondary brain tumors results from metastasis of other malignancy outside of nerve system. Glioma and astrocytoma are two most common types of primary malicious brain tumors. Patients diagnosed with malignant brain tumor in general have poor prognosis. For example,Glioblastoma multiforme (GBM), the most common and lethal type of adult primary brain tumors, have a median survival of less than 11 months.[1] Current stand of care for metastasis neoplasm is surgical resection followed by radiation and chemotherapy.[2] High remission of tumor growth and inevitable death despite extensive use of standard therapeutic interventions sets the demand for new treatment.

Gene therapy offers the promises of argumenting traditional cancer regimes as well as enabling new approaches. Therapeutic genes can act as cytotoxic agent, angiogenesis antagonist, immune response stimulator or restore endogenous anti-tumor regulation by supplementing the mutated genes that underlying certain cancer types.[3] However, the therapeutic effect of gene therapy is hindered by the inefficient delivery to site of action. Neural stem cells kick in as a powerful delivery vehicle thanks to their remarkable migratory and tumor- tropic properties.[4]

1. Current Condition

1.1. Clinical challenges

Conventional treatment
surgical resection for brain tumor

Conventional treatment can only prolong survival by a few months before relapse and is associated with toxicity that hamper quality of life. The dismal of current treatments is due to both intrinsic and extrinsic characteristics of brain tumors. Cancerous cells in brain are highly infiltrative and located perivascularly and parineuronally, they can easily migrate though whiter matter tracts, blood vessels and CSF. Heterogeneity is another nature of the tumors cells, as the tumor mass harbor same location can generate different sections of microenvironment in proximity. Such properties also causes difficulty in identifying tumor margin markers.[5]The discovery of cancer stem cells provided another explanation for cancer recurrence as they can escape therapies that targeting rapid dividing cells by slowing down cell proliferation or even temporarily exit cell cycle. Extrinsically, brain is immune privilege as it is shielded by blood brain barrier (BBB). Much of the blood, which contains both immune responsive cells and anti-tumor agents, will not reach brain tumor bed. Novel strategies have been developed to boost therapeutic efficacy includes anti-angiogenesis, tumor cells sensitization to chemo- and radio- therapies and co-administration of immune stimulator etc.[2]

1.2. Experimental challenges

Since 1930s, scientists have introduced rodent glioma cell lines to study brain tumor. They inject mutagens such as alkylating agents intracranially (adults) or intravenously (prenatal) to induce cancer formation in animals.[6] However the resulted mutation range is more widespread than human cases. The human glioma cell lines, in particular GBM-derived cells are of great interest. Yet they can only be studied in immune-suppressed animals. There are debates regards to whether human glioma cell lines are adequate to represent original GBM.[7] Thanks to the identification of genetic etiologies of gliomas, germline gliomagenesis-based transgenic and knock-out models became popular models to study brain tumors. In many cases, combination of overexpressed mutated genes and gene knock-outs has been necessary to induce brain tumors. Some of the common combinations are overexpression of v12H-Ras and p53 knockouts, overexpression of v-src and NF1 knockouts.[8] Despite the rapid development of new models, certain challenges remain: tumors can only been induced in young animals; there is variability in the genetic background; tumor penetrance varies from generation to generation etc.[6] Another standard use of murine tumor models is via tumor transplantation. Such syngeneic GBM models allow study of immunologic response to tumor cells.[9-10]

2. Traditional Gene Therapy

The lack of cure and ideal disease management of malignant brain tumors set the need for effective and low-toxicity regimes. Since 1990s, the study of gene therapy has thrived in a large extend. First gene therapy clinical trial was conducted in 1989 in melanoma patients, promising results are soon reported in 1990 for two children with Severe combined immunodeficiency (SCID) using retrovirus encoding adenosine deaminase (ADA) gene.[11-12] Numerous therapeutic strategies for gene therapy have been developed base on the complex nature of cancers. They can be grouped into cytotoxic approach and immunologic approach.

2.1. Cytotoxic approach

Ideal gene therapies for brain tumor are assessed by three factors: the kind of anti-tumoric information/transgenes be transmitted into tumor cells, the anti-cancer signals selectively target tumor cells and the effective transfection of cytotoxic signal to all tumor cells.[13]

Tumor-specific

One way of restricting cytotoxic agent expression is the use of tumor- specific promoter such as hTERT. So the administrated vectors that encoded with therapeutic transgene will lead to p53-upregulated modulator of apoptosis (PUMA) in tumor cells expressed hTERT only.[14]
Targeting receptors that are expressed exclusively on tumor cells is another strategy to constrain the pro-apoptotic effect to tumor cells. IL-13Ralpha2 receptor, for example, is found upregulated in most human GBM tumor cells but not in normal brain cells. Therefore constructed adenoviral vectors that encoded with mutated human IL-13Ralpha2 can fuse to highly cytotoxic proteins to direct cell death in tumor only. Mutated human IL-4 that binds to physiological receptor are also added to protect normal brain cells from mhIL-13Ralpha2.[15]
Hypoxia is a typical condition in tumors resulted from insufficient blood supply. Such tumor-medicated microenvironment can be exploited for selective anti-tumor effect as well. Bax, another pro-apoptotic molecules can be encoded into vectors that get transfected into GBM cells and is facilitated by hypoxia responsive elements.[16]

Correction of genetic defects

Tumor suppressor genes (such as p53) and oncogenes (such as Bcl-2, c-myc and ras) are the two major gene groups that responsible for carcinogenesis and progression. Treatments that combine adenovirus transfected wild type p53 gene with chemotherapy and radiotherapy have shown to inhibit tumor growth and regression. To inhibit oncogene biological activity, antisense oligonucleotides and antigene olgonnucleotides are used to target RNA and DNA respectively. Down regulation of tumor growth is fulfilled via inference with transcription and translation of protein synthesis.[17]
What’s more, RNA interference as a new gene therapy approach in cancer is gaining more and more attention in use of short interfering RNAs.[18]

Prodrug activation

Perhaps the most documented gene therapy application is suicide gene therapy. It starts with conversion of non-toxic prodrugs into cytotoxic agents in tumor cells upon introduction of non-mammalian or mammalian enzymes. One of the earliest prodrug activation system is herpes simplex virus thymidine kinase (HSVst)/Ganciclovir(GCV) system. HSVst is a recombinant fene product made by herpes virus and viral enzyme. Thymidine kinases can metabolite GCV into antiviral drug by phosphorylation. The phosphorylated GCV will complete the second part of suicide gene therapy via a mechanism called bystander effect. Such mechanism takes advantage of intercellular communication like gap junctions or apoptotic vesicles of tumor cells and spread toxic agents through tumor mass. Other similar systems like cytosine deaminase (CD)/5-fluorocytosine(5-FC), cytochrome P450/cyclophosphamide are also suitable candidates for suicide gene therapy. Combination of multiple prodrug activation systems and immunologic activation have shown heightened therapeutic effect than either one of them alone.[19-21]

2.2. Immunologic approach

Example of Immunologic Gene Therapy
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The effects of adenoviral mediated TK/Flt3L gene therapy [10]

As mentioned previously, brain tissues are largely immunological privilege in partly due to blood brain barrier. Unique physiologic features, for example: the lack of dendritic cells and lymphatic drainage, secretion of anti-inflammatory mediator (TGF-B and NO) by cells like infiltrating microglia serve as protective mechanisms for vital organs in the context of immunologic attack in normal individuals. However, these intrinsic factors likewise result in weaker tumorigenic detection and rejection inside the brain, namely brain tumors. The idea of immunotherapy is elevating natural immunologic response, more specifically, T-Cell mediated immunologic response towards malignant cells.[13]

Activation of antigen presenting cells

One of the common strategies in immunological gene therapy is to prime immune response. Deliver dendritic cells (DC) to the site of action when the antigen is present in tumor microenvironment. This can be achieved by co-activation of TK/GCV system and fms-like tyrosine kinase-3 ligand (Flt3L) expression. The former causes tumor cell death and release of antigen while latter is a potent DC growth factor that operates to attract infiltrating DCs to tumor microenvironment. The tumor specific response relies on the interaction between Toll-like receptor 2 expressed on DCs and the tumor derived ligand, high-mobility-group box 1 (HMGB1).[10]

Immune-stimulatory cytokines

IL-2 and IL-12 are cytokines that crucial for cytotoxic T cell argumentation. Latter is an important facet to adaptive immune response. Several research groups have managed to develop and apply recombinant vaccinia virus express those cytokines to suppress tumor growth in rat glioma models.[22]
Tumor necrosis factor-alpha (TNFa) and Interferon-gamma are another set of immune-stimulatory cytokines. However they promote immune response by facilitating cell growth and migration of immune cells including DCs, neutral killer cells, B cells, T cells etc.23 TNFa also thought to involve in anti-angiogenesis and direct necrosis in neoplastic cells..[24-25]

3. Human stem cell as cargo

3.1. Potential of human stem cells

Tumor tropism

Tumor Tropism
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Multipotent mesenchymal stromal cells (MSCs) in vitro and within gliomas. [27]

The first documentation of tumor-tropism property of human stem cells came out a decade ago. Mesenchymal stem cells (MSCs) grafted to rats bearing syngeneic gliomas were found to migrate to and distribute throughout the tumor mass.[26] Further experiments revealed the extended feature of MSCs to outline invasive glioma extension and distant tumor microsatellites but avoid normal brain tissue. Certain research groups believed that such attraction is mediated by tumor microenvironment as latter provides a permissive niche for MSC survival.[28] The routes of administration for therapeutic MSCs includes intravenous, intra-arterial and intraperitoneal injections, and intracerebral, intratcerebroventricular, and intratumoral implantation. Upon systematic injection, MSCs are trapped in both normal and pathologic tissues initially. Although the amount of MSCs gradually decreased in normal tissue, intratumoral injection reminds the most popular route of administration for both safety and efficacy reason.[27]

Mechanism

There are several suggested mechanisms for the tumor-tropism of MSCs. Early reports indicated it is mediated by platelet-derived growth factor (PDGF)-BB, stromal-derived factor-1 and epidermal growth factor.[29] Other investigators believed that glioma-produced angiogenic cytokines are involved in MSC migration within tumors. This postulation is reinforced by pericyte-like property of MSCs implied by recent data.[30] Xenografted MSCs cells are found to act like supportive cells and interact with endothelial cells to maintain vessel integrity and facilitate blood flow. Furthermore, they express pericyte makers, such as neuron-glia 2, PDGFRB, and alpha-smooth muscle actin.[31] Such property has significant clinical relevance, as it can be exploited to maximize therapeutic benefit, specifically targeting the most rapidly proliferating parts of the tumor and channel for delivery of anti-tumor agents. MSCs are also found to involved in wound healing, suggesting inflammatory mediators may play a role in MSCs migration. However, no specific MSC sub-population markers identified to data, and more studies regard to heterogeneity of MSC is needed to further understand the mechanism of MSCs.[32]

3.2. Advantages

Powerful vehicle

MSCs can serve as a vehicle for delivery of prodrug activation enzymes, cytokines, oncolytic viruses etc. The advanced distributive potential can significantly increase the efficiency of suicide gene therapy even under systematic administration. MSCs loaded with conditionally replicating oncolytic viruses can escape from immune attack before it reaches the site of action. The association of tumor angiogenesis and implanted MSCs allow targeting of both angiogenesis and rapid dividing tumor cells. Last but not least, the proliferative capability of stem cells can prolong therapeutic effect of carried anti-tumor agents. These inherent properties grant huge therapeutic advantage for MSCs vehicles in comparison with viruses, antibodies, Nanoparticles, and lipomas. [27]

In vivo monitoring

MRI-based imaging allows non-invasive in vivo monitoring of transgene expression and vector distribution. Cationic polymersomes labeled MSCs can be detected by MRI machines up to 6 months post-injection for as few as 1000 cells.[33] Bioluminescence imaging can track the migration of luciderase gene-transduced NSC non-invasively. Yet it generates relative poor resolution due to limited light penetration trough tissues as compared to MRI. Positron emission tomography is also used to study hMSCs. Not like MRI imaging, it calls for radiolabeling or cells pre-grafting and has time limitation due to the short half-life of radio-ligands.[27],[34]

3.3. Risks

MSCs are mostly cultured in fetal calf serum (FCS)-containing medium, both in experiment setting and clinical use. FCS is a very complex supplement constituting a mixture of undefined proteins, growth factors, hormones, amino acids etc and even infectious molecules like prions. Alternatives such as platelet lysate or serum-free MSC culture system have been investigated for MSC culturing. Another risk is the possibility of MSCs transform into malignant cells following long-term ex vivo culturing.[35] Tightly regulated culture time and clonal expansion of MSC is needed particularly for clinical use. What’s more, the immunosuppressive property of MSC can compete with the antitumor immune response that induced by the therapeutic agents it carried. Recent studies have shown heterogeneous influence on immune effector cells elicited by MSCs.[36] Finally, certain type of MSCs, such as bone marrow-derived cells are capable of fusion with tumor cells, which may lead to increased tumor growth, drug resistance and metastasis.[37]

4. Implication in cancer stem cells

The most common malignant brain tumor in children is Medulloblastoma, which is a primitive neuroectodermal tumor of cerebellum.[38] In comparison to malignant brain tumors in adults, most of the pediatric brain tumor patients survive > 5 years under relative same stand of care (Surgery followed by radiation and chemotherapy). However, impair cognitive function and depressive symptoms has been correlated with cancer treatment.[39] One suggestion of such outcome is the involvement of neural stem cells that responsible for adult neurogenesis. Patients treated for malignant brain tumor also has higher risk of endocrine deficits and develop secondary tumors later in life.[36]

4.1. Origin and propagation of brain tumor

Lineage Development
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Normal cells that give rise to brain tumors.[36]

The cell of origin for cancer is defined as the normal cells from which that a tumor arises. The cell of origin for human tumors used to be determined based on expression markers that associated with specific cell type. However, the understanding of cell of origin for brain tumor is very limited in part due to the possibility that cell marker expression may be altered during transformation; in part due to the heterogeneity of brain tumor mass that multiple sets of cell markers can present on tumor masses which appear as one entity anatomically. An elucidated cell origin for brain tumor is benefit for developing suitable experimental model as well as therapies. Thanks to the advancement of genetic modifying technologies in the past two decades. Scientists have identified several genetic aetiologies that corresponding to different brain types, and the number is still increasing.[36]

4.2. Analogies between stem cells and cancer

The concept of cancer stem cells first came on the stage at 1997 when researches shown subpopulation of cells from acute myeloid leukemia that graft to immune-deficient was capable of generating tumor mass.[40] Phenotypically, cancer stem cells resemble stem cells to a large extend as they both responsible for initiation, proliferation and maintenance of respected lineages. Many scientists believed that cancer stem cells are immune to conventional chemotherapy and radiation. It believed to associate with its ability of entering dormant stage. Caner stem cells are thought to play a huge ruole in high relapse of brain tumor post-surgery. It is not surprised to found that cancer stem cells share some of the physiological signals and pathways with stem cells for self-renewal and regeneration. For example, targeting of Shh and Wnt pathways, which are important proliferation and differentiation signals during development, have shown to inhibit pathogenesis of brain cancers.[41] Other growth factors or genes that coded for neural stem cell growth mediators have also been indicated in pathogenesis of various types of brain tumors.[36]

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