Correction of feline lipoprotein lipase deficiency with adeno-associated virus serotype 1-mediated gene transfer of the lipoprotein lipase S447X beneficial mutation.

Human lipoprotein lipase (hLPL) deficiency, for which there currently exists no adequate treatment, leads to excessive plasma triglycerides (TGs), recurrent abdominal pain, and life-threatening pancreatitis. We have shown that a single intramuscular administration of adeno-associated virus (AAV) serotype 1 vector, encoding the human LPL(S447X) variant, results in complete, long-term normalization of dyslipidemia in LPL(/) mice. As a prelude to gene therapy for human LPL deficiency, we tested the efficacy of AAV1-LPL(S447X) in LPL(/) cats, which demonstrate hypertriglyceridemia (plasma TGs, >10,000 mg/dl) and clinical symptoms similar to LPL deficiency in humans, including pancreatitis. Male LPL(/) cats were injected intramuscularly with saline or AAV1-LPL(S447X) (1 x 10(11)-1.7 x 10(12) genome copies [GC]/kg), combined with oral doses of cyclophosphamide (0-200 mg/m(2) per week) to inhibit an immune response against hLPL. Within 3-7 days after administration of >or=5 x 10(11) GC of AAV1-LPL(S447X) per kilogram, the visible plasma lipemia was completely resolved and plasma TG levels were reduced by >99% to normal levels (10-20 mg/dl); intermediate efficacy (95% reduction) was achieved with 1 x 10(11) GC/kg. Injection in two sites, greatly limiting the amount of transduced muscle, was sufficient to completely correct the dyslipidemia. By varying the dose per site, linear LPL expression was demonstrated over a wide range of local doses (4 x 10(10)-1 x 10(12) GC/site). However, efficacy was transient, because of an anti-hLPL immune response blunting LPL expression. The level and duration of efficacy were significantly improved with cyclophosphamide immunosuppression. We conclude that AAV1-mediated delivery of LPL(S447X) in muscle is an effective means to correct the hypertriglyceridemia associated with feline LPL deficiency.

Rescue and sprouting of motoneurons following ventral root avulsion and reimplantation combined with intraspinal adeno-associated viral vector-mediated expression of glial cell line-derived neurotrophic factor or brain-derived neurotrophic factor.

Following avulsion of a spinal ventral root, motoneurons that project through the avulsed root are axotomized. Avulsion between, for example, L2 and L6 leads to denervation of hind limb muscles. Reimplantation of an avulsed root directed to the motoneuron pool resulted in re-ingrowth of some motor axons. However, most motoneurons display retrograde atrophy and subsequently die. Two neurotrophic factors, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), promote the survival of motoneurons after injury. The long-term delivery of these neurotrophic factors to the motoneurons in the ventral horn of the spinal cord is problematic. One strategy to improve the outcome of the neurosurgical reinsertion of the ventral root following avulsion would involve gene transfer with adeno-associated viral (AAV) vectors encoding these neurotrophic factors near the denervated motoneuron pool. Here, we show that AAV-mediated overexpression of GDNF and BDNF in the spinal cord persisted for at least 16 weeks. At both 1 and 4 months post-lesion AAV-BDNF- and -GDNF-treated animals showed an increased survival of motoneurons, the effect being more prominent at 1 month. AAV vector-mediated overexpression of neurotrophins also promoted the formation of a network of motoneuron fibers in the ventral horn at the avulsed side, but motoneurons failed to extent axons into the reinserted L4 root towards the sciatic nerve nor to improve functional recovery of the hind limbs. This suggests that high levels of neurotrophic factors in the ventral horn promote sprouting, but prevent directional growth of axons of a higher number of surviving motoneurons into the implanted root.

Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors.

BACKGROUND: Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. RESULTS: Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. CONCLUSION: AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

Adeno-associated viral vector-mediated gene transfer of brain-derived neurotrophic factor reverses atrophy of rubrospinal neurons following both acute and chronic spinal cord injury.

Rubrospinal neurons (RSNs) undergo marked atrophy after cervical axotomy. This progressive atrophy may impair the regenerative capacity of RSNs in response to repair strategies that are targeted to promote rubrospinal tract regeneration. Here, we investigated whether we could achieve long-term rescue of RSNs from lesion-induced atrophy by adeno-associated viral (AAV) vector-mediated gene transfer of brain-derived neurotrophic factor (BDNF). We show for the first time that AAV vectors can be used for the persistent transduction of highly atrophic neurons in the red nucleus (RN) for up to 18 months after injury. Furthermore, BDNF gene transfer into the RN following spinal axotomy resulted in counteraction of atrophy in both the acute and chronic stage after injury. These novel findings demonstrate that a gene therapeutic approach can be used to reverse atrophy of lesioned CNS neurons for an extended period of time.

Cells in human postmortem brain tissue slices remain alive for several weeks in culture.

Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue slices obtained by autopsy within 8 h after death can be maintained in vitro for extended periods (up to 78 days) and can be manipulated experimentally. We report for the first time that 1) neurons and glia in such cultures could be induced to express the reporter gene LacZ after transduction with adeno-associated viral vectors and 2) cytochrome oxidase activity could be enhanced by the addition of pyruvate to the medium. These slice cultures offer new opportunities to study the cellular and molecular mechanisms of neurological and psychiatric diseases and new therapeutic strategies.