Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders.
Viral vectors carrying suicide genes such as diphtheria toxin, Pseudomonas exotoxin, or barnase are very useful tools in cancer gene therapy and cell ablation research. However, such viral vectors are extremely difficult to produce due to the fact that trace amounts of the toxin will kill any cells used for viral vector production. To overcome this obstacle, we inserted mammalian introns that are not recognized by insect cells to break up the open reading frames (ORFs) of the toxic genes and successfully produced at normal levels of baculoviral and adeno-associated viral (AAV) vectors carrying these toxic genes. Once these viral vectors were used to infect mammalian cells, the introns were spliced out and toxic proteins expressed to kill the target cells.
Here we evaluated the utility of MRI to monitor intrathecal infusions in nonhuman primates. Adeno-associated virus (AAV) spiked with gadoteridol, a gadolinium-based MRI contrast agent, enabled real-time visualization of infusions delivered either via cerebromedullary cistern, lumbar, cerebromedullary and lumbar, or intracerebroventricular infusion. The kinetics of vector clearance from the cerebrospinal fluid (CSF) were analyzed. Our results highlight the value of MRI in optimizing the delivery of infusate into CSF. In particular, MRI revealed differential patterns of infusate distribution depending on the route of delivery. Gadoteridol coverage analysis showed that cerebellomedullary cistern delivery was a reliable and effective route of injection, achieving broad infusate distribution in the brain and spinal cord, and was even greater when combined with lumbar injection. In contrast, intracerebroventricular injection resulted in strong cortical coverage but little spinal distribution. Lumbar injection alone led to the distribution of MRI contrast agent mainly in the spinal cord with little cortical coverage, but this delivery route was unreliable. Similarly, vector clearance analysis showed differences between different routes of delivery. Overall, our data support the value of monitoring CSF injections to dissect different patterns of gadoteridol distribution based on the route of intrathecal administration.
Two mammalian introns, the human growth hormone intron and the Simian virus 40 large T antigen intron, were inserted into the coding sequences of diphtheria toxin fragment A (DT-A) and barnase (Bar), respectively, to disrupt their open-reading frames (ORFs). Expression of these two toxic proteins were totally abolished, which enabled the production of normal levels of recombinant baculoviral and adeno-associated viral (AAV) vectors in insect cells. When these viral vectors were introduced into mammalian cells, the introns were spliced out and the toxic proteins were expressed, which resulted in apoptosis in mammalian cells. This is the first report to show that viral vectors harboring toxin genes can be produced at normal levels by exploiting the intron-splicing mechanism of insect cells. Furthermore, viral vectors carrying the DT-A gene under control of tumor-specific promoters were able to exert tumor-specific cell killing. This novel method to produce viral vectors harboring toxic genes under control of tumor-specific promoter offers a powerful tool for further research, as well as for the development of toxin-based suicide gene therapy drugs.Molecular Therapy - Nucleic Acids (2012) 1, e57; doi:10.1038/mtna.2012.48; published online 27 November 2012.
Adeno-associated virus (AAV) is one of the most promising vectors for gene therapy. There are several ways of producing AAV vectors but large-scale production of this vector remains a major challenge. Virovek developed a novel method of expressing the AAV Rep and Cap genes in insect cells mediated by intron-splicing mechanism and producing AAV vectors with these Rep and Cap sequences containing the artificial intron. The recombinant baculoviruses harboring these artificial intron-containing Rep and Cap sequences are very stable and the AAV vectors produced in insect cells with these recombinant baculoviruses are very infectious.
An artificial intron containing the insect cell polyhedrin (polh) promoter was designed, constructed, and inserted into the adeno-associated virus (AAV) Rep and Cap coding sequences to express the Rep and Cap proteins, respectively. The artificial intron was spliced out and full-length Rep78 or VP1 proteins were expressed from the insect promoters located upstream of their respective AUG start codons. The polh promoter located inside the artificial intron was functional, expressed the Rep52 or VP2/VP3 proteins located downstream of the artificial intron, and overlapped with the Rep78 or VP1 proteins. This is the first report that an artificial intron containing an insect cell promoter can be inserted into a coding sequence to express genes with overlapping open-reading frames (ORFs). A method was also established for AAV vector production in insect cells with these intron-containing Rep and Cap coding sequences, and the vectors produced thereby were infectious. These intron-containing AAV Rep and Cap coding sequences were very stable in recombinant baculoviruses and showed no apparent loss of protein expression even after five consecutive amplifications of the plaque-purified recombinant baculoviruses. This newly established AAV production method should prove to be a useful tool for large-scale AAV vector production.
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