16 research outputs found
Aurothiolates enhance the replication of Semliki Forest virus in the CNS and the exocrine pancreas
AAV2-mediated in vivo immune gene therapy of solid tumours
Abstract Background Many strategies have been adopted to unleash the potential of gene therapy for cancer, involving a wide range of therapeutic genes delivered by various methods. Immune therapy has become one of the major strategies adopted for cancer gene therapy and seeks to stimulate the immune system to target tumour antigens. In this study, the feasibility of AAV2 mediated immunotherapy of growing tumours was examined, in isolation and combined with anti-angiogenic therapy. Methods Immune-competent Balb/C or C57 mice bearing subcutaneous JBS fibrosarcoma or Lewis Lung Carcinoma (LLC) tumour xenografts respectively were treated by intra-tumoural administration of AAV2 vector encoding the immune up-regulating cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and the co-stimulatory molecule B7-1 to subcutaneous tumours, either alone or in combination with intra-muscular (IM) delivery of AAV2 vector encoding Nk4 14 days prior to tumour induction. Tumour growth and survival was monitored for all animals. Cured animals were re-challenged with tumourigenic doses of the original tumour type. In vivo cytotoxicity assays were used to investigate establishment of cell-mediated responses in treated animals. Results AAV2-mediated GM-CSF, B7-1 treatment resulted in a significant reduction in tumour growth and an increase in survival in both tumour models. Cured animals were resistant to re-challenge, and induction of T cell mediated anti-tumour responses were demonstrated. Adoptive transfer of splenocytes to naïve animals prevented tumour establishment. Systemic production of Nk4 induced by intra-muscular (IM) delivery of Nk4 significantly reduced subcutaneous tumour growth. However, combination of Nk4 treatment with GM-CSF, B7-1 therapy reduced the efficacy of the immune therapy. Conclusions Overall, this study demonstrates the potential for in vivo AAV2 mediated immune gene therapy, and provides data on the inter-relationship between tumour vasculature and immune cell recruitment
Validation of a Lysis Buffer Containing 4 M Guanidinium Thiocyanate (GITC)/ Triton X-100 for Extraction of SARS-CoV-2 RNA for COVID-19 Testing: Comparison of Formulated Lysis Buffers Containing 4 to 6 M GITC, Roche External Lysis Buffer and Qiagen RTL Lysis Buffer
The COVID-19 pandemic has resulted in increased need for diagnostic testing using reverse transcriptase real-time PCR (RT-PCR). An exponential increase in demand has resulted in a shortage of numerous reagents in particular those associated with the lysis buffer required to extract the viral RNA. Herein, we describe a rapid collective effort by hospital laboratory scientists, academic researchers and the biopharma industry to generate a validated lysis buffer. We have formulated a 4M Guanidinium thiocyanate (GITC)/ Triton X-100 Lysis buffer which provides comparable results with the recommended reagents. This buffer will ease the burden on hospital labs in their heroic efforts diagnose a large population of patients
Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA.
Cystic Fibrosis is an autosomal recessive disorder caused by mutations in the CFTR gene. CRISPR mediated, template-dependent homology-directed gene editing has been used to correct the most common mutation, c.1521_1523delCTT / p.Phe508del (F508del) which affects ~70% of individuals, but the efficiency was relatively low. Here, we describe a high efficiency strategy for editing of three different rare CFTR mutations which together account for about 3% of individuals with Cystic Fibrosis. The mutations cause aberrant splicing of CFTR mRNA due to the creation of cryptic splice signals that result in the formation of pseudoexons containing premature stop codons c.1679+1634A>G (1811+1.6kbA>G) and c.3718-2477C>T (3849+10kbC>T), or an out-of-frame 5' extension to an existing exon c.3140-26A>G (3272-26A>G). We designed pairs of Cas9 guide RNAs to create targeted double-stranded breaks in CFTR either side of each mutation which resulted in high efficiency excision of the target genomic regions via non-homologous end-joining repair. When evaluated in a mini-gene splicing assay, we showed that targeted excision restored normal splicing for all three mutations. This approach could be used to correct aberrant splicing signals or remove disruptive transcription regulatory motifs caused by deep-intronic mutations in a range of other genetic disorders
Viral vectors in cancer immunotherapy: which vector for which strategy?
Gene therapy involves the transfer of genetic information to a target cell to facilitate the production of therapeutic proteins and is now a realistic prospect as a cancer treatment. Gene transfer may be achieved through the use of both viral and non-viral delivery methods and the role of this method in the gene therapy of cancer has been demonstrated. Viruses represent an attractive vehicle for cancer gene therapy due to their high efficiency of gene delivery. Many viruses can mediate long term gene expression, while some are also capable of infecting both dividing and non-dividing cells. Given the broadly differing capabilities of various viral vectors, it is imperative that the functionality of the virus meets the requirements of the specific treatment. A number of immunogene therapy strategies have been undertaken, utilising a range of viral vectors, and studies carried out in animal models and patients have demonstrated the therapeutic potential of viral vectors to carry genes to cancer cells and induce anti-tumour immune responses. This review critically discusses the advances in the viral vector mediated delivery of immunostimulatory molecules directly to tumour cells, the use of viral vectors to modify tumour cells, the creation of whole cell vaccines and the direct delivery of tumour antigens in animal models and clinical trials, specifically in the context of the suitability of vector types for specific strategies
Mini-gene analysis of c.3140-26A>G mutation.
<p>a) Schematic of pMG-in19-WT plasmid comprising a contiguous ~2.0 kb region of <i>CFTR</i> containing part of intron 18, exon 19, all of intron 19, exon 20 and part of intron 20 cloned between the 5’ and 3’ exons of the pSPL3 vector. b) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in19-WT. c) Schematic of pMG-in19-A>G mutation which differs from pMG-in19-WT by the A>G change shown with dotted arrow. The A>G change extends exon 20 by 25 bp which shifts the reading frame and eventually results in-frame stop codon (TGA). The triangles indicate target sites of Cas9/gRNAs encoded by pTandem-in19 vector. Arrows above DNA represent in19Ufw and pspSEQrv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in19-A>G. e) Schematic of pMG-in19-A>G mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in19-A>G measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in19-A>G only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in19-A>G and pTandem-in12. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in19-A>G and pTandem-in19 vector.</p
Mini-gene analysis of c.3718-2477C>T mutation.
<p>a) Schematic of pMG-in22-WT plasmid comprising a contiguous ~3.2 kb region of <i>CFTR</i> containing part of intron 22, exon 23, and part of intron 23 cloned between the 5’ and 3’ exons of the pSPL3 vector. b) Electropherogram analysis of RT-PCR products (filled peaks) generated from HEK293T cells transfected with pMG-in22-WT. c) Schematic of pMG-in22-C>T mutation which differs from pMG-in22-WT by the C>T change shown with dotted arrow. The C>T change creates a pseudo exon (Ψ-EX) which contains an in-frame stop codon (TAA). The triangles flanking the Ψ-EX indicate target sites of Cas9/gRNAs encoded by pTandem-in22 vector. Arrows above DNA represent pspSEQfw and in22Drv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in22-C>T. e) Schematic of pMG-in22-C>T mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in22-C>T measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in22-C>T only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in22-C>T and pTandem-in22. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in22-C>T and pTandem-in22 vector.</p