To date, the majority of CF patients receive symptomatic treatment. Gene therapy offers the potential to cure CF in mutation-independent manner. Here, we evaluate rAAV2/5 gene therapy for CF in human ...intestinal organoids and a ΔF508 mouse model. First, we studied the longevity of gene expression after a single administration of a rAAV2/5-Fluc to mouse airways. This resulted in relatively stable gene expression up to 15 months with only a 4-fold decrease in bioluminescent signal in lungs. Due to the rAAV size limit, we used a truncated CFTR missing a portion of the R-domain, CFTRΔR (Ostedgaard et al., PNAS, 2005). First, we evaluated its functionality in a physiological relevant and highly translational organoid model. These data demonstrate that the therapeutic vector rAAV-CFTRΔR is functional and that the ion transport-induced organoid swelling is CFTR-specific. Indeed, despite the expression cassette being at the limit of the rAAV packaging capacity, intact genomes are incorporated into vector particles, that allow efficient second strand DNA synthesis and transgene expression. Finally, we administered rAAV2/5-CFTRΔR to ΔF508 mice by nasal instillation. 2–4 weeks later, we demonstrated a response to low-chloride and forskolin perfusion in 6 out of 8 mice by nasal potential differences, indicating restoration of chloride transport across nasal mucosa. In conclusion, we obtained sustained reporter gene expression in murine airways and demonstrated restoration of the CF phenotype in CF organoids and a CF mouse model. Our results underscore the therapeutic potential of rAAV2/5-CFTRΔR for CF gene therapy opening new avenues towards a definitive cure for all CF patients.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinson’s disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing ...GTPase and kinase domains. LRRK2 can autophosphorylate
in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.
Dopamine dysregulation syndrome shares some core behavioral features with psychostimulant addiction, suggesting that dopamine replacement therapy can acquire psychostimulantlike properties in some ...patients with Parkinson disease (PD). We here report strong experimental evidence supporting this hypothesis in an α‐synuclein rat model of PD. Although levodopa had no effect in controls, it acquired 2 prominent psychostimulantlike properties in Parkinsonian rats: (1) it produced intense reward on its own and in parallel (2) decreased interest in other nondrug reward. These 2 effects may combine to explain the addictive use of levodopa after loss of midbrain dopamine neurons in some PD patients. Ann Neurol 2013;74:140–144
Gene discovery and gene therapy call for advanced technologies to reliably assess gene expression; efficient coupling of gene expression to the expression of reporter genes is critical. Various ...noninvasive molecular imaging modalities have emerged to track biological processes in animal models. Here, we evaluate various strategies to link transgene expression with that of an (imaging) reporter gene. Using lentiviral vectors containing internal ribosomal entry sites (IRES), 2A-like peptides, or a bidirectional promoter, we compared their ability to ensure efficient coexpression of multiple reporter genes. Although the encephalomyocarditis virus (EMCV) IRES yielded functional bicistronic vectors, the expression level of the reporter downstream of IRES was consistently lower than that of the upstream transgene. Interestingly, peptide 2A constructs performed best in vitro and in vivo, providing effective noninvasive follow-up of transgene expression and having reporter gene expression levels in line with that of the single reporter constructs. The intrinsic "cleavage" property of the peptide 2A sequences allows each protein to be produced at proportional levels, opening ample possibilities for functional genomics and future gene therapeutic applications. Last, using various peptide 2A sequences, we engineered the triple reporter LV-3R (i.e., eGFP, fLuc, HSV1-sr39tk), enabling efficient multimodality readouts in vivo.
Transgenic mice overexpressing different forms of amyloid precursor protein (APP), i.e. wild type or clinical mutants, displayed an essentially comparable early phenotype in terms of behavior, ...differential glutamatergic
responses, deficits in maintenance of long term potentiation, and premature death. The cognitive impairment, demonstrated
in F1 hybrids of the different APP transgenic lines, was significantly different from nontransgenic littermates as early as
3 months of age. Biochemical analysis of secreted and membrane-bound APP, C-terminal âstubs,â and Aβ(40) and Aβ(42) peptides
in brain indicated that no single intermediate can be responsible for the complex of phenotypic dysfunctions. As expected,
the Aβ(42) levels were most prominent in APP/London transgenic mice and correlated directly with the formation of amyloid
plaques in older mice of this line. Plaques were associated with immunoreactivity for hyperphosphorylated tau, eventually
signaling some form of tau pathology. In conclusion, the different APP transgenic mouse lines studied display cognitive deficits
and phenotypic traits early in life that dissociated in time from the formation of amyloid plaques and will be good models
for both early and late neuropathological and clinical aspects of Alzheimerâs disease.
Mutations in the homologous presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of familial Alzheimer's disease. Although PS1 function and dysfunction have been ...extensively studied, little is known about the function of PS2 in vivo. To delineate the relationships of PS2 and PS1 activities and whether PS2 mutations involve gain or loss of function, we generated PS2 homozygous deficient (-/-) and PS1/PS2 double homozygous deficient mice. In contrast to PS1-/-mice, PS2-/-mice are viable and fertile and develop only mild pulmonary fibrosis and hemorrhage with age. Absence of PS2 does not detectably alter processing of amyloid precursor protein and has little or no effect on physiologically important apoptotic processes, indicating that Alzheimer's disease-causing mutations in PS2, as in PS1, result in gain of function. Although PS1+/-PS2-/-mice survive in relatively good health, complete deletion of both PS2 and PS1 genes causes a phenotype closely resembling full Notch-1 deficiency. These results demonstrate in vivo that PS1 and PS2 have partially overlapping functions and that PS1 is essential and PS2 is redundant for normal Notch signaling during mammalian embryological development.
RNA interference (RNAi) is an evolutionarily conserved mechanism of posttranscriptional gene-specific silencing. For in vivo applications, RNAi has been hampered until recently by inefficient ...delivery methods and by the transient nature of the gene suppression. Lentiviral vectors (LVs) hold great promise for gene therapeutic applications, pharmaceutical target validation, and functional genomics because stable gene transfer is mediated both in dividing and nondividing cells. We have used a lentiviral vector-based system for RNAi. We produced human immunodeficiency virus type 1-derived LVs encoding a short hairpin RNA specific for enhanced green fluorescent protein (EGFP) mRNA that were capable of inhibiting EGFP expression in mammalian cells. EGFP knockdown persisted after multiple passages of the cells. Of particular interest, our RNAi LVs were equally effective in suppression and prevention of EGFP expression after stereotactic injection in adult mouse brain. Therefore, we believe that the use of LVs for stable RNAi in brain will become a powerful aid to probe gene function in vivo and for gene therapy of diseases of the central nervous system.
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