Delphine Bohl

Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts.

We investigated tetracycline regulation of gene expression in an experimental model relevant to gene therapy. Mouse primary myogenic cells were engineered for doxycycline-inducible and skeletal muscle-specific expression of the mouse erythropoietin (Epo) cDNA by using two retrovirus vectors. Gene expression increased 200 fold in response to both myogenic cell differentiation and doxycycline stimulation. After transplantation of transduced cells into mouse skeletal muscles, Epo secretion could be iteratively switched on and off over a five-month period, depending on the presence or the absence of doxycycline in the drinking water. We conclude that tetracycline regulation provides a suitable control system for adjusting the delivery of therapeutic proteins from engineered tissues over long periods of time.

Lack of an Immune Response against the Tetracycline-Dependent Transactivator Correlates with Long-Term Doxycycline-Regulated Transgene Expression in Nonhuman Primates after Intramuscular Injection of Recombinant Adeno-Associated Virus

ABSTRACT We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design.

Blockade of the insulin-like growth factor I receptor in the choroid plexus originates Alzheimer's-like neuropathology in rodents: New cues into the human disease?

The possibility that perturbed insulin/insulin-like growth factor I (IGF-I) signalling is involved in development of late-onset forms of Alzheimers disease (AD) is gaining increasing attention. We recently reported that circulating IGF-I participates in brain amyloid beta (Abeta) clearance by modulating choroid plexus function. We now present evidence that blockade of the IGF-I receptor in the choroid plexus originates changes in brain that are reminiscent of those found in AD. In rodents, IGF-I receptor impairment led to brain amyloidosis, cognitive disturbance, and hyperphosphorylated tau deposits together with other changes found in Alzheimers disease such as gliosis and synaptic protein loss. While these disturbances were mostly corrected by restoring receptor function, blockade of the IGF-I receptor exacerbated AD-like pathology in old mutant mice already affected of brain amyloidosis and cognitive derangement. These findings may provide new cues into the causes of late-onset Alzheimers disease in humans giving credence to the notion that an abnormal age-associated decline in IGF-I input to the choroid plexus may contribute to development of AD in genetically prone subjects.

Control of Erythropoietin Secretion by Doxycycline or Mifepristone in Mice Bearing Polymer-Encapsulated Engineered Cells

Cell encapsulation offers a safe and manufacturable method for the systemic delivery of therapeutic proteins from genetically engineered cells. However, control of dose delivery remains a major issue with regard to clinical application. We generated populations of immortalized murine NIH 3T3 fibroblasts that secrete mouse erythropoietin (Epo) in response to stimulation by doxycycline or mifepristone. Engineered cells were introduced into AN69 hollow fibers, which were implanted in the peritoneal cavity or recipient mice. Animals receiving doxycycline or mifepristone showed stable polyhemia and increased serum Epo concentrations over a 6-month observation period, whereas animals not receiving the inducer drug had normal hematocrits. Epo secretion could be switched on and off, depending on the presence of doxycycline in the drinking water. In contrast, polyhemia was hardly reversible after subcutaneous injections of mifepristone. These data show that a permanent and regulated systemic delivery of a therapeutic protein can be obtained by the in vivo implantation of engineered allogeneic cells immunoprotected in membrane polymers.

Alsin/Rac1 signaling controls survival and growth of spinal motoneurons.

Recessive mutations in alsin, a guanine‐nucleotide exchange factor for the GTPases Rab5 and Rac1, cause juvenile amyotrophic lateral sclerosis (ALS2) and related motoneuron disorders. Alsin function in motoneurons remained unclear because alsin knock‐out mice do not develop overt signs of motoneuron degeneration.

Selective uptake and sustained expression of AAV vectors following subcutaneous delivery

Recombinant adeno‐associated viral (rAAV) vectors are capable of long‐term expression of secreted and intracellular proteins following delivery to muscle, liver, and the central nervous system. In this study, we have evaluated subcutaneous injection of rAAV encoding a variety of transgenes as an alternative route of administration for the systemic delivery of therapeutic proteins.

Modeling neuronal defects associated with a lysosomal disorder using patient-derived induced pluripotent stem cells

By providing access to affected neurons, human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease, defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system, causing relentless progression toward severe mental retardation. Partially digested proteoglycans, which affect fibroblast growth factor signaling, accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages, patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures, undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions, whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together, these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues, which possibly affect Golgi organization, cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development, once HS proteoglycan accumulation becomes prominent in the affected child brain.

Neural Progenitors Derived From Human Induced Pluripotent Stem Cells Survive and Differentiate Upon Transplantation Into a Rat Model of Amyotrophic Lateral Sclerosis

Human induced pluripotent stem cells (iPSCs) offer hope for personalized regenerative cell therapy in amyotrophic lateral sclerosis (ALS). We analyzed the fate of human iPSC‐derived neural progenitors transplanted into the spinal cord of wild‐type and transgenic rats carrying a human mutated SOD1(G93A) gene. The aim was to follow survival and differentiation of human neural progenitors until day 60 post‐transplantation in two different in vivo environments, one being ALS‐like. iPSC‐derived neural progenitors efficiently engrafted in the adult spinal cord and survived at high numbers. Different neural progenitor, astroglial, and neuronal markers indicated that, over time, the transplanted nestin‐positive cells differentiated into cells displaying a neuronal phenotype in both wild‐type and transgenic SOD1 rats. Although a transient microglial phenotype was detected at day 15, astroglial staining was negative in engrafted cells from day 1 to day 60. At day 30, differentiation toward a neuronal phenotype was identified, which was further established at day 60 by the expression of the neuronal marker MAP2. A specification process into motoneuron‐like structures was evidenced in the ventral horns in both wild‐type and SOD1 rats. Our results demonstrate proof‐of‐principle of survival and differentiation of human iPSC‐derived neural progenitors in in vivo ALS environment, offering perspectives for the use of iPSC‐based therapy in ALS.

Forced expression of the motor neuron determinant HB9 in neural stem cells affects neurogenesis.

In contrast to mouse embryonic stem cells and in spite of overlapping gene expression profiles, neural stem cells (NSCs) isolated from the embryonic spinal cord do not respond to physiological morphogenetic stimuli provided by Sonic hedgehog and retinoic acid and do not generate motor neurons upon differentiation. Transcription factors expressed in motor neuron progenitors during embryogenesis include Pax6, Ngn2, Nkx6.1 and Olig2, whose expression precedes that of factors specifying motor neuron fate, including HB9, Islet1 and LIM3. We showed that all these factors were present in neural progenitors derived from mouse ES cells, whereas NSCs derived from the rat embryonic spinal cord expressed neither HB9 nor Islet1 and contained low levels of Nkx6.1 and LIM3. We constructed a lentivirus vector to express HB9 and GFP in NSCs and examined the consequences of HB9 expression on other transcription factors and cell differentiation. Compared to cell expressing GFP alone, NSCs expressing GFP and HB9 cycled less rapidly, downregulated Pax6 and Ngn2 mRNA levels, produced higher proportions of neurons in vitro and lower numbers of neurons after transplantation in the spinal cord of recipient rats. Oligodendrocytic and astrocytic differentiations were not affected. HB9 expressing NSCs did not express Islet1 or upregulate LIM3. They neither responded to Sonic hedgehog and retinoic acid nor produced cholinergic neurons. We concluded that forced HB9 expression affected neurogenesis but was not sufficient to confer motor neuron fate to NSCs.

Modeling amyotrophic lateral sclerosis in pure human iPSc-derived motor neurons isolated by a novel FACS double selection technique

Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease. Human motor neurons generated from induced pluripotent stem cells (iPSc) offer new perspectives for disease modeling and drug testing in ALS. In standard iPSc-derived cultures, however, the two major phenotypic alterations of ALS--degeneration of motor neuron cell bodies and axons--are often obscured by cell body clustering, extensive axon criss-crossing and presence of unwanted cell types. Here, we succeeded in isolating 100% pure and standardized human motor neurons by a novel FACS double selection based on a p75(NTR) surface epitope and an HB9::RFP lentivirus reporter. The p75(NTR)/HB9::RFP motor neurons survive and grow well without forming clusters or entangled axons, are electrically excitable, contain ALS-relevant motor neuron subtypes and form functional connections with co-cultured myotubes. Importantly, they undergo rapid and massive cell death and axon degeneration in response to mutant SOD1 astrocytes. These data demonstrate the potential of FACS-isolated human iPSc-derived motor neurons for improved disease modeling and drug testing in ALS and related motor neuron diseases.