Motoko Yamamoto

Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken β-Globin Locus HS4 Insulator Element

Hematopoietic cell gene therapy using retroviral vectors has achieved success in clinical trials. However, safety issues regarding vector insertional mutagenesis have emerged. In two different trials, vector insertion resulted in the transcriptional activation of proto-oncogenes. One strategy for potentially diminishing vector insertional mutagenesis is through the use of self-inactivating lentiviral vectors containing the 1.2-kb insulator element derived from the chicken beta-globin locus. However, use of this element can dramatically decrease both vector titer and transgene expression, thereby compromising its practical use. Here, we studied lentiviral vectors containing either the full-length 1.2-kb insulator or the smaller 0.25-kb core element in both orientations in the partially deleted long-terminal repeat. We show that use of the 0.25-kb core insulator rescued vector titer by alleviating a postentry block to reverse transcription associated with the 1.2-kb element. In addition, in an orientation-dependent manner, the 0.25-kb core element significantly increased transgene expression from an internal promoter due to improved transcriptional termination. This element also demonstrated barrier activity, reducing variability of expression due to position effects. As it is known that the 0.25-kb core insulator has enhancer-blocking activity, this particular insulated lentiviral vector design may be useful for clinical application.Hematopoietic cell gene therapy using retroviral vectors has achieved success in clinical trials. However, safety issues regarding vector insertional mutagenesis have emerged. In two different trials, vector insertion resulted in the transcriptional activation of proto-oncogenes. One strategy for potentially diminishing vector insertional mutagenesis is through the use of self-inactivating lentiviral vectors containing the 1.2-kb insulator element derived from the chicken β-globin locus. However, use of this element can dramatically decrease both vector titer and transgene expression, thereby compromising its practical use. Here, we studied lentiviral vectors containing either the full-length 1.2-kb insulator or the smaller 0.25-kb core element in both orientations in the partially deleted long-terminal repeat. We show that use of the 0.25-kb core insulator rescued vector titer by alleviating a postentry block to reverse transcription associated with the 1.2-kb element. In addition, in an orientation-dependent manner, the 0.25-kb core element significantly increased transgene expression from an internal promoter due to improved transcriptional termination. This element also demonstrated barrier activity, reducing variability of expression due to position effects. As it is known that the 0.25-kb core insulator has enhancer-blocking activity, this particular insulated lentiviral vector design may be useful for clinical application.

Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors.

Central nervous system (CNS) disorders are important targets for gene therapy; however, delivery of therapeutic proteins and/or genes to the brain remains a major challenge due to the difficulty of efficiently delivering viral vectors across the blood-brain barrier (BBB). In the present work, we tested the ability of several single-stranded adeno-associated viral (ssAAV) serotypes to deliver transgenes to the brain and spinal cord in neonatal mice. We injected ssAAV vectors encoding GFP (serotype-1, -8, -9 and -10: 1.5×10(11) vector genomes each) into the jugular vein of neonatal mice and assessed GFP expression immunohistochemically. Strong GFP signals were detected in both the brain and spinal cord after injection of any of these serotypes. ssAAV serotype-9 mediated gene transfer was the most efficient. GFP expression was detected throughout the brain, including the cortex, cerebellum, olfactory bulb and brainstem and was sustained for at least 18months. Immunohistochemical staining showed that the GFP signals were detected in GFAP positive astrocytes, NeuN positive neurons, and Calbindin positive purkinje cells. Our data suggest that systemic neonatal injection of ssAAV is an effective strategy for delivering transgenes to target neuronal systems that are not accessible to viral vectors in adult animals. These vectors should prove highly useful for efficient and long-term overexpression or downregulation of genes in CNS and spinal cord and could be a useful means of treating genetic neurological diseases.

Global diffuse distribution in the brain and efficient gene delivery to the dorsal root ganglia by intrathecal injection of adeno-associated viral vector serotype 1

The success of gene therapy for inherited neurodegenerative diseases such as metachromatic leukodystrophy (MLD) depends on the development of efficient gene delivery throughout the brain guarded by the blood–brain barrier and achieves distribution of the deficient enzyme throughout the brain. Direct injection of viral vector into the brain parenchyma is too invasive and may not be sufficient to treat the entire brain. As an alternative approach, we examined the feasibility of intrathecal (IT) injection of adeno‐associated viral vector serotype 1 (AAV1).

IL-1ra versus IL-1 Levels in Prostatic Fluid from Prostatitis Patients

Recent papers reported that the balance between the production of IL-1ra and IL-1 probably influences the regulation of host responses, the severity and prolongation of the inflammatory reaction in some diseases. Therefore, in our continuing investigation to clarify the significance of leukocytosis and its prolongation in prostatic fluid from prostatitis patients, we investigated whether low levels of IL-1ra versus IL-1β secreted in prostatic fluid were the cause of prolonged prostatitis, especially nonbacterial prostatitis (NBP). As a result of the present study, we concluded that a low level of IL-1ra in relation to that of IL-1 secreted in prostatic fluid is unlikely to cause prolongation of NBP for the following reasons: (1) IL-1β was detected in 5 of 10 cases (50.0%), but was slightly elevated in only 2 cases (20.0%) at 14 and 17 pg/ml; (2) the average IL-1ra level was not statistically low compared with that in prostatic fluid from acute bacterial prostatitis (ABP) patients who were cured promptly with antibiotics, and (3) in 5 cases of NBP in which IL-1β was detected, the average IL-1ra/IL-1β ratio was 118 which was comparable to or even higher than that in 3 ABP and 14 acute bacterial cystitis cases in which IL-1β was detected and the ratios were 40 and 88, respectively.

Long-term correction of biochemical and neurological abnormalities in MLD mice model by neonatal systemic injection of an AAV serotype 9 vector

As both the immune system and the blood–brain barrier (BBB) are likely to be developmentally immature in the perinatal period, neonatal gene transfer may be useful for the treatment of lysosomal storage disease (LSD) with neurological involvements such as metachromatic leukodystrophy (MLD). In this experiment, we examined the feasibility of single-strand adeno-associated viral serotype-9 (ssAAV9)-mediated systemic neonatal gene therapy of MLD mice. ssAAV9 vector expressing human arylsulfatase A (ASA) and green fluorescent protein (GFP) (ssAAV9/ASA) was injected into the jugular vein of newborn MLD mice. High levels of ASA expression were observed in the muscle and heart for at least 15 months. ASA was continuously secreted into plasma without development of antibodies against ASA. Global gene transfer into the brain and spinal cord (SC), across the BBB, and long-term ASA expression in the central nervous system were detected in treated mice. Significant inhibition of the accumulation of sulfatide (Sulf) in the brain and cervical SC was confirmed by Alcian blue staining and biochemical analysis of the Sulf content. In a behavior test, treated mice showed a greater ability to traverse narrow balance beams than untreated mice. These data clearly demonstrate that MLD mice model can be effectively treated through neonatal systemic injection of ssAAV9/ASA.

Association between single nucleotide polymorphisms in the hMSH3 gene and sporadic colon cancer with microsatellite instability

AbstractThe association between three single nucleotide polymorphisms (SNPs) in the hMSH3 gene and sporadic colon cancer with microsatellite instability (MSI) was analyzed. Of the three SNPs observed in this population, SNPs at residues 235 and 693 were novel, while that at residue 3133 was previously described. The SNPs at residues 235 and 3133 caused amino acid substitutions, V79I and T1045A, respectively. We analyzed the allele frequencies of the three SNPs in samples from 19 patients with sporadic colon cancer with MSI and 90 healthy controls. We found that the V79 allele frequency was significantly higher in the tumor samples than in controls. In addition, the frequency of the G693 allele showed a higher trend in the tumor samples than in controls. These results indicated that some SNPs in the hMSH3 gene were associated with colon cancer with MSI.

The chicken hypersensitivity site 4 core insulator blocks promoter interference in lentiviral vectors.

Lentiviral vectors, including double internal promoters, can be used to express two transgenes in a single vector construct; however, transcriptional activities from double internal promoters are often inhibited by promoter interference. To determine whether the chicken hypersensitivity site 4 insulator (cHS4) could block promoter interference, lentiviral vectors including an MSCV-U3 promoter (Mp) and an EF1α promoter (Ep) were generated, and transgene expression was evaluated among transduced cells. In the Ep-Mp configuration, transcriptional activity from Mp was much lower, while Mp-Ep had similar transcription levels from both promoters. The cHS4 core insulator increased expression levels from Mp in HeLa cells, hematopoietic cell lines, and mouse peripheral blood cells following hematopoietic stem cell transplantation transduced with the Mp-Ep configured vector. This blocking function was mainly mediated by barrier activity regions in the insulator but not by CCCTC-binding factor (CTCF) binding sites. Cytosine-phosphate-guanine (CpG) methylation did not contribute to this barrier activity. In summary, combining the cHS4 insulator in double promoter vectors can improve transgene expression levels in various cell lines and mouse hematopoietic repopulating cells. These findings are useful for developing hematopoietic stem cell gene therapy.

Enzyme replacement in the CSF to treat metachromatic leukodystrophy in mouse model using single intracerebroventricular injection of self-complementary AAV1 vector

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a functional deficiency in human arylsulfatase A (hASA). We recently reported that ependymal cells and the choroid plexus are selectively transduced by intracerebroventricular (ICV) injection of adeno-associated virus serotype 1 (AAV1) vector and serve as a biological reservoir for the secretion of lysosomal enzymes into the cerebrospinal fluid (CSF). In the present study, we examined the feasibility of this AAV-mediated gene therapy to treat MLD model mice. Preliminary experiments showed that the hASA level in the CSF after ICV injection of self-complementary (sc) AAV1 was much higher than in mice injected with single-stranded AAV1 or scAAV9. However, when 18-week-old MLD mice were treated with ICV injection of scAAV1, the concentration of hASA in the CSF gradually decreased and was not detectable at 12 weeks after injection, probably due to the development of anti-hASA antibodies. As a result, the sulfatide levels in brain tissues of treated MLD mice were only slightly reduced compared with those of untreated MLD mice. These results suggest that this approach is potentially promising for treating MLD, but that controlling the immune response appears to be crucial for long-term expression of therapeutic proteins in the CSF.

160. Successful Treatment of Neonatal Metachromatic Leukodystrophy Model Mice by Low Dose of Self-Complementary AAV Type9 Vector Expressing ASA

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease (LSD) caused by a functional deficiency of the lysosomal enzyme arylsulfatase A (ASA) and characterized by severe neurological symptoms due to widespread and progressive demyelination in the both central and peripheral nervous systems. Enzyme replacement therapy (ERT) has been applied to treat certain types of LSDs, but correction of neurological abnormalities is usually hampered by the blood brain barrier (BBB). We have previously shown that MLD mice can be treated by intravenous (IV) injection of classical single-stranded AAV type 9 vector expressing ASA (ssAAV9/ASA) for neonate and self-complementary AAV9/ASA (scAAV9/ASA) for adult. To develop the clinical MLD gene therapy, it is important to decrease the amount of viral vectors because of safety. In this study, we evaluated the therapeutic potential of scAAV9/ ASA-mediated systemic transduction of neonatal MLD mice at 10 times lower dose (2 × 1011 v.g./body) compared to the treatable dose of the ssAAV9/ASA (2 × 1012 v.g./body). We generated both ssAAV9/ASA and scAAV9/ASA to intravenously inject then into the neonatal MLD mice (n=6-9). ELISA analysis showed that sustained high-level expression of ASA was detected in the brain and spinal cord of the scAAV9/ASA-treated mice (cerebral cortex, 2.1 times; cerebellum, 2 times; spinal cord, 5.5 times) compared to the ssAAV9/ASA for more than one year. Furthermore, in the behavior test, both scAAV9/ASA and ssAAV9/ASA-treated mice showed a significant improvement in their ability to traverse narrow balance beams, as compared to the non-treated MLD mice (latency; 7.2±0.9, 9.3±1.1 vs. 16.6±0.8 sec, P<0.05: slips; 3.4±0.8, 3.6±0.6 vs. 7.3±1.8 times, P<0.05). These data clearly demonstrate that MLD model mice can be treated by systemic neonatal transduction with low dose of scAAV9/ASA. Therefore, neonatal gene therapy would be an important option for parents faced with the prenatal diagnosis of a genetically affected child. Since ten times lower dose of scAAV9/ASA is enough to treat the neonatal MLD mice compared to ssAAV9/ASA, transduction strategy to use scAAV9/ASA should be safe and more practical for the clinical MLD gene therapy.