Cell and gene therapy: a snapshot of investor perspectives.

In a collaborative effort between the Commercialization Committee of the International Society for Cell & Gene Therapy (ISCT) and Bloomberg Intelligence, a broad survey of the investment community was executed in order to understand investor perceptions of companies that develop cell and gene therapies (CGTs) and gauge the trajectory of future investment. A broad spectrum of investors responded to the survey, including both health care specialists and generalist investors across a wide range of fund sizes and geographies. A majority of survey respondents have limited exposure to CGTs in their health care portfolios today, which highlights the opportunity to increase awareness of this burgeoning field in the investment community. The survey established that clinically significant data are the most important consideration when making an investment in this area, whereas safety concerns were highlighted as the most prominent barrier to making an investment. Challenges with manufacturing and scale-up were also ranked as a significant concern. The majority of investors hold the belief that both autologous and allogeneic cell therapies can co-exist. The detailed findings of this survey will help to provide a foundation for educational content that the ISCT Commercialization Committee can bring forth to further the investment in CGTs through the newly created Investigators to Investors program.

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease.

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.

Knowing versus doing: education and training needs of staff in a chronic care hospital unit for individuals with dementia.

Hospital clinical staff routinely confront challenging behaviors in patients with dementia with limited training in prevention and management. The authors of the current article conducted a survey of staff on a chronic care hospital unit concerning knowledge about dementia, perceived educational needs, and the care environment. The overall mean score for a 27-item knowledge scale was 24.08 (SD = 2.61), reflecting high level of disease knowledge. However, staff indicated a need for more information and skills, specifically for managing behaviors nonpharmacologically (92.3%), enhancing patient safety (89.7%), coping with care challenges (84.2%), and involving patients in activities (81.6%). Although most staff (i.e., nurses [80%] and therapists [86.4%]) believed their care contributed a great deal to patient well-being, approximately 75% reported frustration and being overwhelmed by dementia care. Most reported being hit, bitten, or physically hurt by patients (66.7%), as well as disrespected by families (53.8%). Findings suggest that staff have foundational knowledge but lack the "how-to" or hands-on skills necessary to implement nonpharmacological behavioral management approaches and communicate with families.

Smad4 is critical for self-renewal of hematopoietic stem cells.

Members of the transforming growth factor beta (TGF-beta) superfamily of growth factors have been shown to regulate the in vitro proliferation and maintenance of hematopoietic stem cells (HSCs). Working at a common level of convergence for all TGF-beta superfamily signals, Smad4 is key in orchestrating these effects. The role of Smad4 in HSC function has remained elusive because of the early embryonic lethality of the conventional knockout. We clarify its role by using an inducible model of Smad4 deletion coupled with transplantation experiments. Remarkably, systemic induction of Smad4 deletion through activation of MxCre was incompatible with survival 4 wk after induction because of anemia and histopathological changes in the colonic mucosa. Isolation of Smad4 deletion to the hematopoietic system via several transplantation approaches demonstrated a role for Smad4 in the maintenance of HSC self-renewal and reconstituting capacity, leaving homing potential, viability, and differentiation intact. Furthermore, the observed down-regulation of notch1 and c-myc in Smad4(-/-) primitive cells places Smad4 within a network of genes involved in the regulation HSC renewal.

Mitochondrial loss, dysfunction and altered dynamics in Huntington's disease.

Although a direct causative pathway from the gene mutation to the selective neostriatal neurodegeneration remains unclear in Huntington's disease (HD), one putative pathological mechanism reported to play a prominent role in the pathogenesis of this neurological disorder is mitochondrial dysfunction. We examined mitochondria in preferentially vulnerable striatal calbindin-positive neurons in moderate-to-severe grade HD patients, using antisera against mitochondrial markers of COX2, SOD2 and cytochrome c. Combined calbindin and mitochondrial marker immunofluorescence showed a significant and progressive grade-dependent reduction in the number of mitochondria in spiny striatal neurons, with marked alteration in size. Consistent with mitochondrial loss, there was a reduction in COX2 protein levels using western analysis that corresponded with disease severity. In addition, both mitochondrial transcription factor A, a regulator of mtDNA, and peroxisome proliferator-activated receptor-co-activator gamma-1 alpha, a key transcriptional regulator of energy metabolism and mitochondrial biogenesis, were also significantly reduced with increasing disease severity. Abnormalities in mitochondrial dynamics were observed, showing a significant increase in the fission protein Drp1 and a reduction in the expression of the fusion protein mitofusin 1. Lastly, mitochondrial PCR array profiling in HD caudate nucleus specimens showed increased mRNA expression of proteins involved in mitochondrial localization, membrane translocation and polarization and transport that paralleled mitochondrial derangement. These findings reveal that there are both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion in HD. These findings provide further evidence that mitochondrial dysfunction plays a critical role in the pathogenesis of HD.

Reduced creatine kinase as a central and peripheral biomarker in Huntington's disease.

A major goal of current clinical research in Huntington's disease (HD) has been to identify preclinical and manifest disease biomarkers, as these may improve both diagnosis and the power for therapeutic trials. Although the underlying biochemical alterations and the mechanisms of neuronal degeneration remain unknown, energy metabolism defects in HD have been chronicled for many years. We report that the brain isoenzyme of creatine kinase (CK-BB), an enzyme important in buffering energy stores, was significantly reduced in presymptomatic and manifest disease in brain and blood buffy coat specimens in HD mice and HD patients. Brain CK-BB levels were significantly reduced in R6/2 mice by approximately 18% to approximately 68% from 21 to 91 days of age, while blood CK-BB levels were decreased by approximately 14% to approximately 44% during the same disease duration. Similar findings in CK-BB levels were observed in the 140 CAG mice from 4 to 12 months of age, but not at the earliest time point, 2 months of age. Consistent with the HD mice, there was a grade-dependent loss of brain CK-BB that worsened with disease severity in HD patients from approximately 28% to approximately 63%, as compared to non-diseased control patients. In addition, CK-BB blood buffy coat levels were significantly reduced in both premanifest and symptomatic HD patients by approximately 23% and approximately 39%, respectively. The correlation of CK-BB as a disease biomarker in both CNS and peripheral tissues from HD mice and HD patients may provide a powerful means to assess disease progression and to predict the potential magnitude of therapeutic benefit in this disorder.

Uridine ameliorates the pathological phenotype in transgenic G93A-ALS mice.

There is strong evidence from studies in humans and animal models to suggest the involvement of energy metabolism defects in neurodegenerative diseases. Uridine, a pyrimidine nucleoside, has been suggested to be neuroprotective in neurological disorders by improving bioenergetic effects, increasing ATP levels and enhancing glycolytic energy production. We assessed whether uridine treatment extended survival and improved the behavioral and neuropathological phenotype observed in G93A-ALS mice. In vitro and in vivo pharmacokinetic analyses in mutant SOD models provided optimal dose and assurance that uridine entered the brain. A dose-ranging efficacy trial in G93A mice was performed using survival, body weight, open-field analysis, and neuropathology as outcome measures. Urinary levels of 8-hydroxy-2'-deoxyguanosine, identifying DNA oxidative damage, were measured and used as a pharmacodynamic biomarker. Uridine administration significantly extended survival in a dose-dependent manner in G93A mice, while improving the behavioral and neuropathological phenotype. Uridine increased survival by 17.4%, ameliorated body weight loss, enhanced motor performance, reduced gross lumbar and ventral horn atrophy, attenuated lumbar ventral horn neuronal cell death, and decreased reactive astrogliosis. Consistent with a therapeutic effect, uridine significantly reduced urinary 8-hydroxy-2'-deoxyguanosine in G93A mice. These data suggest that uridine may be a therapeutic candidate in ALS patients.

Endoglin is not critical for hematopoietic stem cell engraftment and reconstitution but regulates adult erythroid development.

Endoglin is a transforming growth factor-beta (TGF-beta) accessory receptor recently identified as being highly expressed on long-term repopulating hematopoietic stem cells (HSC). However, little is known regarding its function in these cells. We have used two complementary approaches toward understanding endoglin's role in HSC biology: one that efficiently knocks down expression via lentiviral-driven short hairpin RNA and another that uses retroviral-mediated overexpression. Altering endoglin expression had functional consequences for hematopoietic progenitors in vitro such that endoglin-suppressed myeloid progenitors (colony-forming unit-granulocyte macrophage) displayed a higher degree of sensitivity to TGF-beta-mediated growth inhibition, whereas endoglin-overexpressing cells were partially resistant. However, transplantation of transduced bone marrow enriched in primitive hematopoietic stem and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to short-term or long-term repopulate recipient marrow. Furthermore, transplantation of cells altered in endoglin expression yielded normal white blood cell proportions and peripheral blood platelets. Interestingly, decreasing endoglin expression increased the clonogenic capacity of early blast-forming unit-erythroid progenitors, whereas overexpression compromised erythroid differentiation at the basophilic erythroblast phase, suggesting a pivotal role for endoglin at key stages of adult erythropoietic development.

Treatment of Chemotherapy-Induced Peripheral Neuropathy in Integrative Oncology: A Survey of Acupuncture and Oriental Medicine Practitioners.

OBJECTIVES: Complementary and alternative medicine is increasingly integrated into cancer care. We sought detail on the treatment of chemotherapy-induced peripheral neuropathy (CIPN) with acupuncture and oriental medicine (AOM) by surveying practitioners at integrative oncology (IO) sites across the United States. DESIGN: Online survey of licensed acupuncturists. SETTING/LOCATION: IO sites in the United States. SUBJECTS: Fifteen licensed acupuncturists who completed the survey between February 2014 and June 2014. OUTCOME MEASURES: Demographics, IO setting characteristics, AOM treatment characteristics, and practitioner-reported outcomes. RESULTS: Respondents reported an average of 31.3 ± 17.2 patients per week, and one-third (10.1 mean; 7.2 standard deviation [SD]) were treated for CIPN. Medical doctors (86.7%) were the most common providers with whom respondents worked. Traditional Chinese medicine style acupuncture was utilized by a majority of respondents (86.7%), and the most commonly used points were local, typically in the hands and feet, such as Ba Feng, Ba Xie, LV3, and LI4. In addition to acupuncture, nutritional advice was the most frequent auxiliary modality provided by respondents (85.7%). On average, respondents provided 12.75 ± 4.17 treatments for CIPN patients, and a majority (53%) reported treating patients once per week. Timing of the treatments relative to chemotherapy infusion was evenly distributed between "1-2 days after infusion" (60%), "at time of infusion" (53.3%), and "1-2 days before infusion" (46.7%). Sixty percent of respondents rated outcomes as "moderately successful with moderate improvement seen." CONCLUSION: This survey provides detail regarding IO sites using acupuncture for CIPN as well as real-world treatment patterns, including common point combinations, visit characteristics, and practitioner-reported outcomes. This information contributes to the emerging evidence on the use of acupuncture to address unmet needs of CIPN patients, and supports the development of best practice guidelines for the treatment of CIPN with acupuncture in the IO setting.

Rehabilitation Needs of the Elderly Patient with Cancer.

Physiatrist taking care of the geriatric patient with cancer should be able to manage an array of conditions that might present from diagnosis throughout completion of treatments and beyond. The elderly cancer population is at greater risk of functional impairments. The physician should anticipate changes in clinical status and must adjust rehabilitation goals accordingly. Treatment options and rehabilitation goals should be tailored to help maximize quality of life in these patients.

Feasibility of the Tailored Activity Program for Hospitalized (TAP-H) Patients With Behavioral Symptoms.

PURPOSE OF THE STUDY: To evaluate feasibility of implementing the Tailored Activity Program for Hospitals (TAP-H) to improve engagement in patients with dementia admitted for behavioral disturbances. DESIGN AND METHODS: TAP-H involves up to 11 in-hospital sessions to develop activities tailored to patient interests and capabilities and train staff/families in their use. Interventionists (occupational therapists) recorded session lengths, patient engagement (N = 20), and staff (N = 4) readiness to use activities. Family interviews (N = 20) identified patient behaviors at admission and satisfaction with TAP-H 1 month postdischarge. A time series design with multiple behavioral observations (63 videotaped sessions) compared affective, verbal, and nonverbal behavioral responses in a standardized activity (baseline) to treatment sessions. RESULTS: Average number of treatment sessions per patient was 8.00 (SD = 2.71, range 3-13). Average time spent per session was 38.18min (SD = 10.01, range 19.09-57.50). Interventionists observed high patient engagement across treatment sessions. Observational data revealed increases in pleasure and positive gestures and decreases in anxiety/anger, negative verbalizations, and negative nonverbal behaviors from baseline to intervention sessions. Staff improved in readiness and families expressed high program satisfaction with 59.4% of activities used at home. IMPLICATIONS: TAP-H represents a unique collaborative care model that integrates facility-based staff in the behavioral treatment of patients with dementia and results in improved affect and reduced negative behaviors. TAP-H can be incorporated into routine hospital care and payment mechanisms. Future efforts should evaluate its effectiveness in reducing inappropriate pharmacologic use and strategies to enhance continued activity use by staff during hospitalization and families following discharge.

Feeder-independent culture systems for human pluripotent stem cells.

The continued success of pluripotent stem cell research is ultimately dependent on access to reliable and defined reagents for the consistent culture and cryopreservation of undifferentiated, pluripotent cells. The development of defined and feeder-independent culture media has provided a platform for greater reproducibility and standardization in this field. Here we provide detailed protocols for the use of mTeSR™1 and TeSR™2 with various cell culture matrices as well as defined cryopreservation protocols for human embryonic and human induced pluripotent stem cells.

Clinimetrics corner: the many faces of selection bias.

Selection bias, also known as susceptibility bias in an intervention study or spectrum bias in a diagnostic accuracy study, is present throughout clinically applicable evidence in various forms. Selection bias implies that the intervention or diagnostic test has been studied in a less representative sample population, which can lead to inflated overall effect sizes and/or inaccurate findings. Within the literature, there are over 40 forms of selection bias that can influence the external validity of results. Recognition of selection bias is essential in the translation of evidence into effective clinical practice. This clinimetrics corner outlines the major biases that readers encounter and discusses key examples regarding pertinent orthopedic and manual therapy literature.

Smad5 is dispensable for adult murine hematopoiesis.

Smad5 is known to transduce intracellular signals from bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-beta (TGF-beta) superfamily and are involved in the regulation of hematopoiesis. Recent findings suggest that BMP4 stimulates proliferation of human primitive hematopoietic progenitors in vitro, while early progenitors from mice deficient in Smad5 display increased self-renewal capacity in murine embryonic hematopoiesis. Here, we evaluate the role of Smad5 in the regulation of hematopoietic stem cell (HSC) fate decisions in adult mice by using an inducible MxCre-mediated conditional knockout model. Surprisingly, analysis of induced animals revealed unperturbed cell numbers and lineage distribution in peripheral blood (PB), bone marrow (BM), and the spleen. Furthermore, phenotypic characterization of the stem cell compartment revealed normal numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells in Smad5(-)(/)(-) BM. When transplanted in a competitive fashion into lethally irradiated primary and secondary recipients, Smad5-deficient BM cells competed normally with wild-type (wt) cells, were able to provide long-term reconstitution for the hosts, and displayed normal lineage distribution. Taken together, Smad5-deficient HSCs from adult mice show unaltered differentiation, proliferation, and repopulating capacity. Therefore, in contrast to its role in embryonic hematopoiesis, Smad5 is dispensable for hematopoiesis in the adult mouse.

A road map toward defining the role of Smad signaling in hematopoietic stem cells.

The transforming growth factor-beta (TGF-beta) superfamily encompasses the ligands and receptors for TGF-beta, bone morphogenic proteins (BMPs), and Activins. Cellular response to ligand is context-dependent and may be controlled by specificity and/or redundancy of expression of these superfamily members. Several pathways within this family have been implicated in the proliferation, differentiation, and renewal of hematopoietic stem cells (HSCs); however, their roles and redundancies at the molecular level are poorly understood in the rare HSC. Here we have characterized the expression of TGF-beta superfamily ligands, receptors, and Smads in murine HSCs and in the Lhx2-hematopoietic progenitor cell (Lhx2-HPC) line. We demonstrate a remarkable likeness between these two cell types with regard to expression of the majority of receptors and Smads necessary for the transduction of signals from TGF-beta, BMP, and Activin. We have also evaluated the response of these two cell types to various ligands in proliferation assays. In this regard, primary cells and the Lhx2-HPC line behave similarly, revealing a suppressive effect of Activin-A that is similar to that of TGF-beta in bulk cultures and no effect of BMP-4 on proliferation. Signaling studies that verify the phosphorylation of Smad2 (Activin and TGF-beta) and Smad1/5 (BMP) confirm cytosolic responses to these ligands. In addition to providing a thorough characterization of TGF-beta superfamily expression in HSCs, our results define the Lhx2-HPC line as an appropriate model for molecular characterization of Smad signaling.

Smad7 promotes self-renewal of hematopoietic stem cells.

The Smad-signaling pathway downstream of the transforming growth factor-beta superfamily of ligands is an evolutionarily conserved signaling circuitry with critical functions in a wide variety of biologic processes. To investigate the role of this pathway in the regulation of hematopoietic stem cells (HSCs), we have blocked Smad signaling by retroviral gene transfer of the inhibitory Smad7 to murine HSCs. We report here that the self-renewal capacity of HSCs is promoted in vivo upon blocking of the entire Smad pathway, as shown by both primary and secondary bone marrow (BM) transplantations. Importantly, HSCs overexpressing Smad7 have an unperturbed differentiation capacity as evidenced by normal contribution to both lymphoid and myeloid cell lineages, suggesting that the Smad pathway regulates self-renewal independently of differentiation. Moreover, phosphorylation of Smads was inhibited in response to ligand stimulation in BM cells, thus verifying impairment of the Smad-signaling cascade in Smad7-overexpressing cells. Taken together, these data reveal an important and previously unappreciated role for the Smad-signaling pathway in the regulation of self-renewal of HSCs in vivo.

Anemia, thrombocytopenia, leukocytosis, extramedullary hematopoiesis, and impaired progenitor function in Pten+/-SHIP-/- mice: a novel model of myelodysplasia.

The myeloproliferative disorder of mice lacking the Src homology 2 (SH2)-containing 5' phosphoinositol phosphatase, SHIP, underscores the need for closely regulating phosphatidylinositol 3-kinase (PI3K) pathway activity, and hence levels of phosphatidylinositol species during hematopoiesis. The role of the 3' phosphoinositol phosphatase Pten in this process is less clear, as its absence leads to embryonic lethality. Despite Pten heterozygosity being associated with a lymphoproliferative disorder, we found no evidence of a hematopoietic defect in Pten(+/-) mice. Since SHIP shares the same substrate (PIP(3)) with Pten, we hypothesized that the former might compensate for Pten haploinsufficiency in the marrow. Thus, we examined the effect of Pten heterozygosity in SHIP(-/-) mice, predicting that further dysregulation of PIP(3) metabolism would exacerbate the pheno-type of the latter. Indeed, compared with SHIP(-/-) mice, Pten(+/-)SHIP(-/-) animals developed a myelodysplastic phenotype characterized by increased hepatosplenomegaly, extramedullary hematopoiesis, anemia, and thrombocytopenia. Consistent with a marrow defect, clonogenic assays demonstrated reductions in committed myeloid and megakaryocytic progenitors in these animals. Providing further evidence of a Pten(+/-)SHIP(-/-) progenitor abnormality, reconstitution of irradiated mice with marrows from these mice led to a marked defect in short-term repopulation of peripheral blood by donor cells. These studies suggest that the regulation of the levels and/or ratios of PI3K-derived phosphoinositol species by these 2 phosphatases is critical to normal hematopoiesis.