In vitro regeneration of Psoralea corylifolia Linn.: influence of polyamines during in vitro shoot development

In Vitro Cellular & Developmental Biology - Plant - A reliable and economically feasible in vitro plant regeneration protocol has been standardized for the Psoralea corylifolia Linn. using...     

Polymer‐coated viral vectors: hybrid nanosystems for gene therapy

The advantages and critical aspects of nanodimensional polymer‐coated viral vector systems potentially applicable for gene delivery are reviewed. Various viral and nonviral vectors have been explored for gene therapy. Viral gene transfer methods, although highly efficient, are limited by their immunogenicity. Nonviral vectors have a lower transfection efficiency as a result of their inability to escape from the endosome. To overcome these drawbacks, novel nanotechnology‐mediated interventions that involve the coating or modification of virus using polymers have emerged as a new paradigm in gene therapy. These alterations not only modify the tropism of the virus, but also reduce their undesirable interactions with the biological system. Also, co‐encapsulation of other therapeutic agents in the polymeric coating may serve to augment the treatment efficacy. The viral particles can aid endosomal escape, as well as nuclear targeting, thereby enhancing the transfection efficiency. The integration of the desirable properties of both viral and nonviral vectors has been found beneficial for gene therapy by enhancing the transduction efficiency and minimizing the immune response. However, it is essential to ensure that these attempts should not compromise on the inherent ability of viruses to target and internalize into the cells and escape the endosomes.     

HIV Risk-Reduction Counseling and Testing on Behavior Change of MSM

HIV and AIDS incidence in China is high among men who have sex with men (MSM) and ours was one of few studies in China to evaluate the role of HIV risk reduction counseling and testing. Respondent-driven sampling (RDS) was used to recruit 430 MSM. Participants were followed up at 6, 12 and 18 months to evaluate behavioral changes after counseling to reduce risk behaviors. At baseline, HIV prevalence was 4.7%, whereas HIV incidence was 5.2 per 100 person-years. The incidence was 3.8 during six to 12 months, and 1.1 during 12 to 18 months. During the study period, the reported unprotected anal intercourse (UAI) significantly decreased from 60.9% to 42.9%. The proportion of participants who had one or no partner significantly increased from 40.9% to 48.0%. The study also found that some risk behaviors decreased between baseline and 12 months, followed by a slight increase between 12 and 18 months. Reductions in UAI can be achieved through counseling and testing, but may wane over time. Future programs should consider HIV risk-reduction counseling and testing for interventions in MSM in China.     

High-Efficiency Transduction of Primary Human Hematopoietic Stem Cells and Erythroid Lineage-Restricted Expression by Optimized AAV6 Serotype Vectors In Vitro and in a Murine Xenograft Model In Vivo

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34⁺ cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34⁺ cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.     

Book Review

To study the variability and to identify the species of Begomovirus associated with yellow mosaic disease of blackgram in Andhra Pradesh, India, infected blackgram samples were collected from six districts belonging to three regions of Andhra Pradesh. The total DNA was isolated by modified CTAB method and amplified with coat protein gene-specific primers (RHA-F and AC abut) resulting in 900?bp gene product. The PCR products were cloned, sequenced and deposited in GenBank. The sequence analysis of six clones showed that the size of amplified CP gene of YMV was 920?bp. Based on nucleotide sequence identity of six isolates representing three regions of Andhra Pradesh, the isolates from Rayalaseema and Telangana region are the same variant of YMV (>99.5% identity) and isolate from coastal Andhra is another variant of YMV (>95.4%) when compared with other region isolates. Comparison of CP gene sequence of YMV-TPT isolate with 27 other isolates in database revealed more than 93.2 and 86.2% identity with MYMIV isolates and less than 80 and 64% identity with MYMY isolates that originate from Indian sub-continent and South-East Asia at nucleotide and amino acid level, respectively. Phylogenetic tree based on CP gene sequences of six isolates with other isolates from GenBank formed unique cluster with MYMIV. Hence the YMV infecting blackgram in Andhra Pradesh is caused by MYMIV rather than MYMY as reported in Tamil Nadu which is adjoining state in southern India.     

Photosynthetic Characteristics in Mulberry during Water Stress and Rewatering

Three-month-old plants of mulberry (Morus alba L. cv. Kanva-2) were subjected to a drought stress by withholding water supply. As the leaf water potential (W) was dropping progressively with the severity of treatment and increasing stress duration, the values of leaf area, dry mass accumulation, total chlorophyll (Chl) content, net photosynthetic rate (PN), stomatal conductance (gs), and transpiration rate (E) were declined. The photosystem 2 (PS2) photochemical efficiency significantly decreased only at a severe stress treatment. The intercellular CO2 concentration (Ci) remained unaltered during a mild stress, yet it increased under moderate and severe stresses. The Ci/gs ratio reflected the mesophyll efficiency during water stress. Rewatering of the plants led to an almost complete recovery of PN, E, and gs, indicating that a short-term stress brings about reversible effects only.     

Infectivity of adeno-associated virus serotypes in mouse testis

Background

Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection.

Results

We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells.

Conclusions

In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.

     

Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo

Background aimsAlthough recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed.MethodsWe evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo.ResultsWe observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo.ConclusionsThese studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.