Mesenchymal stem/stromal cells genetically engineered to produce vascular endothelial growth factor for revascularization in wound healing and ischemic conditions.

Mesenchymal stem/stromal cells (MSCs) may be able to improve ischemic conditions as they can actively seek out areas of low oxygen and secrete proangiogenic factors. In more severe trauma and chronic cases, however, cells alone may not be enough. Therefore, we have combined the stem cell and angiogenic factor approaches to make a more potent therapy. We developed an engineered stem cell therapy product designed to treat critical limb ischemia that could also be used in trauma-induced scarring and fibrosis where additional collateral blood flow is needed following damage to and blockage of the primary vessels. We used MSCs from normal human donor marrow and engineered them to produce high levels of the angiogenic factor vascular endothelial growth factor (VEGF). The MSC/VEGF product has been successfully developed and characterized using good manufacturing practice (GMP)-compliant methods, and we have completed experiments showing that MSC/VEGF significantly increased blood flow in the ischemic limb of immune deficient mice, compared to the saline controls in each study. We also performed safety studies demonstrating that the injected product does not cause harm and that the cells remain around the injection site for more than 1 month after hypoxic preconditioning. An on-demand formulation system for delivery of the product to clinical sites that lack cell processing facilities is in development.

Human Mesenchymal Stem Cells Genetically Engineered to Overexpress Brain-derived Neurotrophic Factor Improve Outcomes in Huntington's Disease Mouse Models.

Huntington's disease (HD) is a fatal degenerative autosomal dominant neuropsychiatric disease that causes neuronal death and is characterized by progressive striatal and then widespread brain atrophy. Brain-derived neurotrophic factor (BDNF) is a lead candidate for the treatment of HD, as it has been shown to prevent cell death and to stimulate the growth and migration of new neurons in the brain in transgenic mouse models. BDNF levels are reduced in HD postmortem human brain. Previous studies have shown efficacy of mesenchymal stem/stromal cells (MSC)/BDNF using murine MSCs, and the present study used human MSCs to advance the therapeutic potential of the MSC/BDNF platform for clinical application. Double-blinded studies were performed to examine the effects of intrastriatally transplanted human MSC/BDNF on disease progression in two strains of immune-suppressed HD transgenic mice: YAC128 and R6/2. MSC/BDNF treatment decreased striatal atrophy in YAC128 mice. MSC/BDNF treatment also significantly reduced anxiety as measured in the open-field assay. Both MSC and MSC/BDNF treatments induced a significant increase in neurogenesis-like activity in R6/2 mice. MSC/BDNF treatment also increased the mean lifespan of the R6/2 mice. Our genetically modified MSC/BDNF cells set a precedent for stem cell-based neurotherapeutics and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis, Alzheimer's disease, and some forms of Parkinson's disease. These cells provide a platform delivery system for future studies involving corrective gene-editing strategies.

Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin.

Huntington's disease (HD) is a fatal, autosomal dominant neurodegenerative disorder caused by an expanded trinucleotide (CAG) repeat in exon 1 of the huntingtin gene (Htt). This expansion creates a toxic polyglutamine tract in the huntingtin protein (HTT). Currently, there is no treatment for either the progression or prevention of the disease. RNA interference (RNAi) technology has shown promise in transgenic mouse models of HD by reducing expression of mutant HTT and slowing disease progression. The advancement of RNAi therapies to human clinical trials is hampered by problems delivering RNAi to affected neurons in a robust and sustainable manner. Mesenchymal stem cells (MSC) have demonstrated a strong safety profile in both completed and numerous ongoing clinical trials. MSC exhibit a number of innate therapeutic effects, such as immune system modulation, homing to injury, and cytokine release into damaged microenvironments. The ability of MSC to transfer larger molecules and even organelles suggested their potential usefulness as delivery vehicles for therapeutic RNA inhibition. In a series of model systems we have found evidence that MSC can transfer RNAi targeting both reporter genes and mutant huntingtin in neural cell lines. MSC expressing shRNA antisense to GFP were found to decrease expression of GFP in SH-SY5Y cells after co-culture when assayed by flow cytometry. Additionally MSC expressing shRNA antisense to HTT were able to decrease levels of mutant HTT expressed in both U87 and SH-SY5Y target cells when assayed by Western blot and densitometry. These results are encouraging for expanding the therapeutic abilities of both RNAi and MSC for future treatments of Huntington's disease.

Predicting Membrane-Active Peptide Dynamics in Fluidic Lipid Membranes.

Understanding the interactions between peptides and lipid membranes could not only accelerate the development of antimicrobial peptides as treatments for infections but also be applied to finding targeted therapies for cancer and other diseases. However, designing biophysical experiments to study molecular interactions between flexible peptides and fluidic lipid membranes has been an ongoing challenge. Recently, with hardware advances, algorithm improvements, and more accurate parameterizations (i.e., force fields), all-atom molecular dynamics (MD) simulations have been used as a "computational microscope" to investigate the molecular interactions and mechanisms of membrane-active peptides in cell membranes (Chen et al., Curr Opin Struct Biol 61:160-166, 2020; Ulmschneider and Ulmschneider, Acc Chem Res 51(5):1106-1116, 2018; Dror et al., Annu Rev Biophys 41:429-452, 2012). In this chapter, we describe how to utilize MD simulations to predict and study peptide dynamics and how to validate the simulations by circular dichroism, intrinsic fluorescent probe, membrane leakage assay, electrical impedance, and isothermal titration calorimetry. Experimentally validated MD simulations open a new route towards peptide design starting from sequence and structure and leading to desirable functions.

Synthetic molecular evolution of antimicrobial peptides.

As we learn more about how peptide structure and activity are related, we anticipate that antimicrobial peptides will be engineered to have strong potency and distinct functions and that synthetic peptides will have new biomedical applications, such as treatments for emerging infectious diseases. As a result of the enormous number of possible amino acid sequences and the low-throughput nature of antimicrobial peptide assays, computational tools for peptide design and optimization are needed for direct experimentation toward obtaining functional sequences. Recent developments in computational tools have improved peptide design, saving labor, reagents, costs, and time. At the same time, improvements in peptide synthesis and experimental platforms continue to reduce the cost and increase the throughput of peptide-drug screening. In this review, we discuss the current methods of peptide design and engineering, including in silico methods and peptide synthesis and screening, and highlight areas of potential improvement.

Distress, quality of life and disability in treatment-seeking young adults with social anxiety disorder.

AIM: This study aimed to: (a) examine whether treatment-seeking young adults with social anxiety disorder (SAD) demonstrate similar degrees of distress, quality of life (QoL) and disability to those with other mental disorders; and (b) investigate the impact of comorbidity, specific comorbid conditions and antidepressants use on distress, QoL and disability in treatment-seeking young adults with SAD. METHODS: A cohort of treatment-seeking young adults (aged 16-45) diagnosed with SAD (N = 298) or other mental health disorders (N = 842; including depression, N = 349; bipolar, N = 141; psychosis, N = 173) completed self-report assessments of distress, QoL and disability. RESULTS: Young adults with SAD showed distress and disability of similar degree to those with most other mental disorders. Specifically, young adults with SAD reported significantly lower QoL than those with major depressive disorder or obsessive-compulsive disorder. Furthermore, young adults with SAD had the most difficulties in getting along with others and the second highest level of distress in comparison to other psychiatric groups. In comparison to antidepressants use, the presence of comorbidity showed a substantial negative influence on these health outcomes, particularly when presenting with comorbid depression or obsessive-compulsive disorder. CONCLUSIONS: Findings highlight significant impairments in young adults seeking treatment for SAD and the important moderating influence of comorbidity. This emphasizes the urgent need for effective management and treatment for its presentation and comorbidities in mental health services targeting young adults.

Autism spectrum disorder: An examination of sex differences in neuropsychological and self-report measures of executive and non-executive cognitive function.

LAY ABSTRACT: Research comparing females and males with a diagnosis of autism suggests that there are sex differences in some characteristics such as behaviour regulation. One area not studied in detail is whether females and males with autism perform differently in tests of cognitive ability. The results of previous research are quite mixed. One explanation may be that some research comparing females and males with autism did not include a neurotypical control group for comparison. As a result, it is not clear whether the sex differences in cognitive ability observed in people with autism are similar to differences between neurotypical males and females. To better understand whether there are unique differences between males and females with autism, it is important to also compare them with neurotypical males and females. In our research, we included a neurotypical group and compared males and females with and without a diagnosis of autism. We found that the sex differences in autism are similar to what we observe in males and females without autism. Our study showed that compared with males, females (with and without autism) do better in assessments of processing speed, cognitive flexibility, verbal learning and memory and semantic fluency. Our results suggest that although females show different cognitive performance to males, these sex differences were not specific to the group with a diagnosis of autism.

Validation of the 21-item Depression, Anxiety, and Stress Scales (DASS-21) in individuals with autism spectrum disorder.

The purpose of the study was to examine the internal consistency and validity of the 21-item Depression Anxiety Stress Scale (DASS-21) in individuals with Autism Spectrum Disorder (ASD) and without intellectual disability (IQ >= 70). Participants (NN = 123) were consecutively recruited from the Brain and Mind Centre in New South Wales, Australia. Internal consistency was determined using Cronbach's alpha. Item-total correlations were evaluated by Pearson's product-moment correlation coefficient. The convergent validity of the DASS-21 was examined by measuring its associations with quality of life and other measures of depression and anxiety. Factorial validity was assessed using confirmatory factor analysis. The DASS-21 demonstrated good internal consistency, adequate convergent validity, and all items exhibited satisfactory item-total correlations. Considering fit indices and factor loadings, the confirmatory factor analysis results provided support for the original 3-factor oblique model consisting of depression, anxiety, and stress factors. The model fit could be further improved with some modifications. Overall, the results indicate that the DASS-21 is a viable self-report screening measure for depression, anxiety, and stress in individuals with ASD and without intellectual disability.

Machine Learning for Differential Diagnosis Between Clinical Conditions With Social Difficulty: Autism Spectrum Disorder, Early Psychosis, and Social Anxiety Disorder.

Differential diagnosis in adult cohorts with social difficulty is confounded by comorbid mental health conditions, common etiologies, and shared phenotypes. Identifying shared and discriminating profiles can facilitate intervention and remediation strategies. The objective of the study was to identify salient features of a composite test battery of cognitive and mood measures using a machine learning paradigm in clinical cohorts with social interaction difficulties. We recruited clinical participants who met standardized diagnostic criteria for autism spectrum disorder (ASD: n = 62), early psychosis (EP: n = 48), or social anxiety disorder (SAD: N = 83) and compared them with a neurotypical comparison group (TYP: N = 43). Using five machine-learning algorithms and repeated cross-validation, we trained and tested classification models using measures of cognitive and executive function, lower- and higher-order social cognition and mood severity. Performance metrics were the area under the curve (AUC) and Brier Scores. Sixteen features successfully differentiated between the groups. The control versus social impairment cohorts (ASD, EP, SAD) were differentiated by social cognition, visuospatial memory and mood measures. Importantly, a distinct profile cluster drawn from social cognition, visual learning, executive function and mood, distinguished the neurodevelopmental cohort (EP and ASD) from the SAD group. The mean AUC range was between 0.891 and 0.916 for social impairment versus control cohorts and, 0.729 to 0.781 for SAD vs neurodevelopmental cohorts. This is the first study that compares an extensive battery of neuropsychological and self-report measures using a machine learning protocol in clinical and neurodevelopmental cohorts characterized by social impairment. Findings are relevant for diagnostic, intervention and remediation strategies for these groups.

In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors.

Serious adverse events in some human gene therapy clinical trials have raised safety concerns when retroviral or lentiviral vectors are used for gene transfer. We evaluated the potential for generating replication-competent retrovirus (RCR) and assessed the risk of occurrence of adverse events in an in vivo system. Human hematopoietic stem and progenitor cells (HSCs) and mesenchymal stem cells (MSCs) transduced with two different Moloney murine leukemia virus (MoMuLV)-based vectors were cotransplanted into a total of 481 immune-deficient mice (that are unable to reject cells that become transformed), and the animals were monitored for 18 months. Animals with any signs of illness were immediately killed, autopsied, and subjected to a range of biosafety studies. There was no detectable evidence of insertional mutagenesis leading to human leukemias or solid tumors in the 18 months during which the animals were studied. In 117 serum samples analyzed by vector rescue assay there was no detectable RCR. An additional 149 mice received HSCs transduced with lentiviral vectors, and were followed for 2-6 months. No vector-associated adverse events were observed, and none of the mice had detectable human immunodeficiency virus (HIV) p24 antigen in their sera. Our in vivo system, therefore, helps to provide an assessment of the risks involved when retroviral or lentiviral vectors are considered for use in clinical gene therapy applications.

Validation of the 36-item and 12-item self-report World Health Organization Disability Assessment Schedule II (WHODAS-II) in individuals with autism spectrum disorder.

The World Health Organization Disability Assessment Schedule II (WHODAS-II) is one of the most widely used generic assessments for measuring disability levels in both clinical and nonclinical populations, with sound psychometrics that is also aligned with the International Classification of Functioning framework. However, its psychometric properties have not been explored extensively in individuals with autism spectrum disorder (ASD). This study examined the psychometric properties of the 36-item and 12-item Self-Report WHODAS-II from 109 individuals diagnosed with ASD and without intellectual disability (IQ ≥ 70). Participants were consecutively recruited from the Brain and Mind Centre in New South Wales, Australia. The WHODAS-II showed adequate internal consistency for all domain scores (α = 0.78-0.97 for 36-item) and for the summary scale (α = 0.95 for 36-item; 0.86 for 12-item). All items also exhibited satisfactory correlations with their respective domain (r = 0.39-0.94 for 36-item) and summary scores (r = 0.42-0.71 for 36-item; 0.42-0.67 for 12-item), except item 4.5 "sexual activity" from the 36-item WHODAS-II (r = 0.19). Concurrent validity was shown by moderate correlations between similar constructs across the WHODAS-II and the World Health Organization Quality of Life BREF (Ps < 0.05). The second-order 7-factor model showed the best fit for the 36-item WHODAS-II, while the second-order 6-factor model demonstrated an acceptable fit for the 12-item WHODAS-II. The model fit could be improved with some modifications. The Schmid-Leiman transformation further confirmed the appropriateness of the second-order factor structure. Overall, the results indicated that the WHODAS-II is a viable generic self-report measure for disability in autistic individuals without ID. Autism Res 2019, 12: 1101-1111. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: The majority of autistic people have a disability with a profound or severe limitation in their core activities. However, there is currently limited research identifying reliable and valid self-report measures for disability in the autistic population. This study examined the psychometric properties of the World Health Organization Disability Assessment Schedule II (WHODAS-II) from 109 autistic individuals without intellectual disability. Our results suggest that the WHODAS-II is a viable generic self-report measure for disability in autistic individuals.

Validation of the social functioning scale: Comparison and evaluation in early psychosis, autism spectrum disorder and social anxiety disorder.

Social functioning is an important component of mental disorders for assessment and treatment. There is no recognised self-report instrument to measure social functioning across disorders where social impairment is significant. The Social Functioning Scale (SFS) has, however, been used to assess social functioning in psychotic disorders, including Schizophrenia and Early Psychosis. The current study investigated the reliability, validity and sensitivity of the SFS in Early Psychosis, Autism Spectrum Disorder (ASD), Social Anxiety Disorder (SAD) and neurotypical control populations. As expected, all clinical groups showed significant impairment on the total and sub-scale scores of the SFS. The SFS showed good internal consistency and concurrent validity for people diagnosed with SAD and Early Psychosis and a similar factors structure was found for these groups. Participants with ASD reported a relatively low internal consistency and poor concurrent validity, as well as a three-component solution. The SFS has also showed a good sensitivity to separate clinical populations and neurotypical controls. This study supports the use of the SFS for those with SAD and Early Psychosis. Lower internal consistency in ASD populations suggests further research in larger samples is required and that the relationship between its scales are likely different to other populations. Alternative scales or significant other reports may be required for adults with ASD.

Self-reported empathy in adults with autism, early psychosis, and social anxiety disorder.

The Empathy Quotient (EQ) self-report questionnaire is used to measure empathy in individuals with clinical conditions that have been associated with social impairments. In this study, older teens and adults with autism spectrum disorder (ASD; N = 60), early psychosis (EP; N = 51) and social anxiety disorder (SAD; N = 71) and neurotypical controls (NT; N = 26) were compared on the cognitive empathy, emotional reactivity and social skills sub-scales of the Empathy Quotient (EQ) measure. All three clinical groups reported lower cognitive empathy than NT controls, and the ASD group reported lower cognitive empathy than EP and SAD groups. The ASD group reported lower emotional reactivity than the SAD group. All three clinical groups reported lower social skills that NT controls. The poor self-rated empathy for the ASD and EP groups generally reflects previous research that found individuals with these conditions perform relatively poorly on certain objective measures of empathy. However, the poor self-rated cognitive empathy and social skills for the SAD group conflicts with previous research that has found that SAD groups perform well on objective measures of empathy. This suggests that both EQ and objective measures should be used to fully assess empathy in clinical groups.

Disability, functioning, and quality of life among treatment-seeking young autistic adults and its relation to depression, anxiety, and stress.

In this study, we consecutively recruited treatment-seeking young autistic adults without intellectual impairment aged 16-30 years who presented to a mental health service and evaluated general health (distress, quality of life, and disability), functioning (work loss days and social functioning), and mood symptoms (depression, anxiety, and stress) in those diagnosed with autism spectrum disorder (n = 96). This group was compared to young adults presenting to the same service with primary mental health disorders (depression, n = 343; bipolar, n = 132; psychosis, n = 166; and anxiety, n = 303). This study also investigated the influence of mood symptoms on general health and functioning in the autism spectrum disorder group. Young autistic adults reported significant general health and functioning impairments that were of similar degree to those presenting with primary mental health disorders. Interestingly, the autistic group also reported similarly high levels of mood symptoms to those with primary depressive and anxiety disorders. In the autistic group, depressive symptoms were strongly associated with distress, quality of life, and work loss days, while stress symptoms were strongly associated with disability. This study highlights further research, and mental health services are required specifically targeting young autistic adults to address their significant unmet needs.

Lentiviral vector transduction of a dominant-negative Rev gene into human CD34+ hematopoietic progenitor cells potently inhibits human immunodeficiency virus-1 replication.

Gene therapy for human immunodeficiency virus (HIV)-1 may be performed by introducing into hematopoietic stem cells genes that inhibit replication of HIV-1 using lentiviral vectors. However, production of lentiviral vectors derived from HIV-1 may be inhibited by the gene being carried to inhibit HIV-1 and these vectors could be mobilized by wild-type HIV-1 infecting transduced cells. This study investigates these problems for the delivery of a dominant-negative rev gene humanized revM10 (huM10) by a lentiviral vector. Although most packaging plasmids suffered inhibition of expression of HIV-1 virion proteins by vectors expressing huM10, the packaging plasmids that expressed the highest levels of HIV-1 virion proteins produced vectors at titers that would be sufficient for clinical applications. The vectors carrying huM10 were used to transduce primary human CD34(+) hematopoietic progenitor cells and yielded high-level transduction without toxicity and conferred potent inhibition of HIV-1. The use of lentiviral vectors with deletion of the enhancers and promoter from the LTR (self-inactivating (SIN) vectors) decreased the frequency of vector mobilization by wild-type HIV-1; SIN vectors carrying huM10 were not mobilized detectably. These studies indicate that lentiviral vectors can be made effective for use in gene therapy for HIV-1.

Effective suicide gene therapy for leukemia in a model of insertional oncogenesis in mice.

The safety of gene therapy using hematopoietic stem cells may be increased by including a suicide gene in the therapeutic vector to eliminate adverse events like insertional oncogenesis while retaining the clinical benefits. We have developed a model of experimental insertional oncogenesis by transducing the murine factor-dependent leukemia cell line Ba/F3 with a bicistronic Moloney murine leukemia virus retroviral vector encoding a murine oncogene (cKit(D814V)) in addition to one of three suicide genes: Herpes simplex virus thymidine kinase (HSV-TK); SR39, an HSV-TK mutant with an increased affinity for the drug substrate Ganciclovir (GCV); or sc39, a splice-corrected version of SR39. Following intravenous challenge with transduced Ba/F3 clones and treatment with GCV, leukemia developed in mice given cells expressing HSV-TK, but not SR39 or sc39. In vitro GCV resistance was observed in heterogeneously transduced Ba/F3 pools at 2.5-14%, and single-nucleotide changes or partial loss of the suicide gene were identified as mechanisms of drug escape. However, GCV treatment resulted in 80-100% survival of mice challenged even with pools of partially resistant Ba/F3 cells expressing SR39 or sc39. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity.

Autism, Early Psychosis, and Social Anxiety Disorder: a transdiagnostic examination of executive function cognitive circuitry and contribution to disability.

The disability burden in clinical cohorts with social impairment is significant, leading to poor functional outcomes. Some of this impairment has been linked to executive dysfunction. In this study, a transdiagnostic approach was taken to identify executive function (EF) processes in young adults that may underpin social impairment and to evaluate their contribution to disability. Comparisons were made between three prominent disorders that are characterized by social impairments, Autism Spectrum Disorder (ASD), Early Psychosis (EP) and Social Anxiety Disorder (SAD), as well as a neurotypically developing group (TYP). We examined whether overall disability could be predicted by neuropsychological and self-report assessments of EF. Our study showed that ASD participants demonstrated impaired performance on most domains of EF compared to the TYP group (mental flexibility, sustained attention and fluency) while the EP group showed impairment on sustained attention and attentional shifting. The SAD participants showed EF impairment on self-report ratings, even though their objective performance was intact. Self-reports of EF explained a significant percentage (17%) of disability in addition to the variance explained by other predictors, and this was particularly important for ASD. This is the first study to compare EF measures across clinical groups of social impairment and suggests unique cognitive-circuitry that underpins disability within groups. Impairments in EF were broad in ASD and predicted disability, EP impairments were specific to attentional processes and SAD impairments likely relate to negative self-monitoring. Self-report, as opposed to performance-based EF, provided best capacity to predict disability. These findings contribute to transdiagnostic circuitry models and intervention strategies.

Dietary exposure of Canadians to perfluorinated carboxylates and perfluorooctane sulfonate via consumption of meat, fish, fast foods, and food items prepared in their packaging.

Human exposure to perfluorinated compounds is a worldwide phenomenon; however, routes of human exposure to these compounds have not been well-characterized. Fifty-four solid food composite samples collected as part of the Canadian Total Diet Study (TDS) were analyzed for perfluorocarboxylates and perfluorooctanesulfonate (PFOS) using a methanol extraction liquid chromatography tandem mass spectrometry method. Foods analyzed included fish and seafood, meat, poultry, frozen entrées, fast food, and microwave popcorn collected from 1992 to 2004 and prepared as for consumption. Nine composites contained detectable levels of perfluorinated compounds-four meat-containing, three fish and shellfish, one fast food, and one microwave popcorn. PFOS and perfluorooctanoate (PFOA) were detected the most frequently; concentrations ranged from 0.5 to 4.5 ng/g. The average dietary intake of total perfluorocarboxylates and PFOS for Canadians was estimated to be 250 ng/day, using results from the 2004 TDS composites. A comparison with intakes of perfluorocarboxylates and PFOS via other routes (air, water, dust, treated carpeting, and apparel) suggested that diet is an important source of these compounds. There was a substantial margin of exposure between the toxicological points of reference and the magnitude of dietary intake of perfluorinated compounds for Canadians >/= 12 years old.

Stable gene transfer to human CD34(+) hematopoietic cells using the Sleeping Beauty transposon.

OBJECTIVE: Methods of gene transfer to hematopoietic stem cells that result in stable integration may provide treatments for many inherited and acquired blood diseases. It has been demonstrated previously that a gene delivery system based on the Sleeping Beauty (SB) transposon can be derived where a plasmid transiently expressing the SB transposase can mediate the stable chromosomal integration of a codelivered second plasmid containing a gene expression unit flanked by the inverted repeats derived from the transposon. METHODS: Plasmid DNA containing the elements required for SB transposition was delivered to hematopoietic cells via electroporation. Integrated transgene (enhanced green fluorescent protein [eGFP]) expression was assessed in vitro and in vivo. RESULTS: In the K562 human hematopoietic cell line, we observed stable expression of eGFP in >60% of cells for over 2 months after electroporation of the two plasmids; in contrast, in control cells either not treated with transposase or exposed to a defective mutant transposase, the level of gene expression had fallen to near background (<0.1%) by 2 weeks. In purified human cord blood CD34(+) progenitor cells, the transposase led to stable gene transfer at levels up to 6% for over 4 weeks, but gene transfer to more primitive nonobese diabetic/severe combined immunodeficient repopulating cells or CD34(+)/CD38(-) in long-term culture was low and electroporation of the cells with plasmid DNA caused significant cell death. CONCLUSION: The long-term stable expression highlights the potential of this transposase-based gene delivery method for ameliorating diseases affecting the hematopoietic system, although further improvements in gene transfer efficacy are needed.

Effects of busulfan dose escalation on engraftment of infant rhesus monkey hematopoietic stem cells after gene marking by a lentiviral vector.

OBJECTIVE: Non-myeloablative cytoreduction is used in clinical hematopoietic stem cell gene therapy trials to increase engraftment of gene-modified cells. We utilized an infant rhesus monkey model to identify an optimal dosage of busulfan that results in efficient long-term gene marking with minimal toxicities. METHODS: Bone marrow (BM) was harvested, followed by a single 2-hour intravenous infusion of busulfan at escalating dosages of 0 to 160 mg/m(2). CD34(+) cells were immunoselected from BM, transduced overnight with a simian immunodeficiency virus-based lentiviral vector carrying a non-expressed marker gene, and injected intravenously 48 hours post-busulfan administration. Pharmacokinetics were assessed, as well as adverse effects and peripheral blood and BM gene marking. RESULTS: Increasing dosages of busulfan resulted in increased area-under-the-curve (AUC) with some variability at each dosage level, suggesting interindividual variation in clearance. Blood chemistries were normal and no adverse effects were observed as a result of busulfan infusion. At 120 and 160 mg/m(2), transient neutropenia and thrombocytopenia were noted but not lymphopenia. Over the 6 months of study posttransplantation, a busulfan dosage-related increase in gene marking was observed ranging from undetectable (no busulfan) up to 0.1% gene-containing cells in animals achieving the highest busulfan AUC. This corresponds to a more than 100-fold increase in gene marking over the busulfan dosage range studied. CONCLUSIONS: These data indicate that increased gene marking of hematopoietic stem cells can be achieved by escalating busulfan dosages from 40 to 160 mg/m(2) without significant toxicity in infant nonhuman primates.