Current Issue : January - March Volume : 2017 Issue Number : 1 Articles : 5 Articles
Objective: Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease\ngene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single\ninverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here\nwe aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems.\nMethods: Oversized (>8 kb) transgene constructs containing ABCA4 coding sequence were packaged\nas truncated fragments <5 kb in size into various AAV serotypes. In vitro transductions with these fAAV vector\npreparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and\nwestern blot techniques.\nResults: Transductions with fAAV vector preparations yielded ABCA4 mRNA, but did not generate detectable\nlevels of protein. Sequencing of the transcript population revealed the presence of full length ABCA4 CDS with\nadditional hybrid ABCA4 variants, indicating truncated transgenes without regions of overlap were joining and forming\nstable hybrid transgenes. In contrast, an ABCA4 overlapping dual vector system (OV) with a defined complementary\nregion generated only full length mRNA transcripts plus detectable ABCA4 protein.\nConclusion: Despite previous success shown with the fAAV approach, the lack of repeatability and identification\nof stable hybrid transcripts capable of protein production suggests there is more refinement required before\nconsidering this approach in a clinical setting....
Background: Synthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene\ntherapy. We recently developed a ZF transcription repressor (ZF-KOX1) able to bind to expanded DNA CAG-repeats in the\nhuntingtin (HTT) gene, which are found in Huntingtonâ��s disease (HD). This ZF acutely repressed mutant HTT expression in\na mouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In the present work, we sought\nto develop a long-term single-injection gene therapy approach in the brain.\nMethod: Since non-self proteins can elicit immune and inflammatory responses, we designed a host-matched analogue\nof ZF-KOX1 (called mZF-KRAB), to treat mice more safely in combination with rAAV vector delivery. We also tested a\nneuron-specific enolase promoter (pNSE), which has been reported as enabling long-term transgene expression, to see\nwhether HTT repression could be observed for up to 6 months after AAV injection in the brain.\nResults: After rAAV vector delivery, we found that non-self proteins induce significant inflammatory responses in the\nbrain, in agreement with previous studies. Specifically, microglial cells were activated at 4 and 6 weeks after treatment\nwith non-host-matched ZF-KOX1 or GFP, respectively, and this was accompanied by a moderate neuronal loss. In\ncontrast, the host-matched mZF-KRAB did not provoke these effects. Nonetheless, we found that using a pCAG promoter\n(CMV early enhancer element and the chicken �²-actin promoter) led to a strong reduction in ZF expression by 6 weeks\nafter injection. We therefore tested a new non-viral promoter to see whether the host-adapted ZF expression could be\nsustained for a longer time. Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat)\nresulted in up to 77 % repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 1010\nvirions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that\nlonger term effects are possible.\nConclusion: Host-adapted ZF-AAV constructs displayed a reduced toxicity and a non-viral pNSE promoter improved\nlong-term ZF protein expression and target gene repression. The optimized constructs presented here have potential for\ntreating HD....
constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients\ndevelop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without\nthe complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer\ninto hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading\nto persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly\nundesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO)\ndelivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in\nHSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1�± promoter generated initially therapeutic levels of FVIII,\nhowever, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In\ncontrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1b�± promoter achieved platelet-specific\nFVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable\ntherapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal\nIO delivery vehicle since FVIII stored in �±-granules of platelets is protected from high-titer anti-FVIII antibodies; and that\neven relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot\nformation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and\npersistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate\nlong-term stable expression of hFVIII in platelets and correct hemophilia phenotype for long term. This approach has\nhigh potential to permanently treat FVIII deficiency with and without pre-existing anti-FVIII antibodies....
Gene therapy protocols require robust and long-term gene expression. For two decades, retrovirus family vectors\nhave offered several attractive properties as stable gene-delivery vehicles. These vectors represent a technology with\nwidespread use in basic biology and translational studies that require persistent gene expression for treatment of\nseveral monogenic diseases. Immunogenicity and insertional mutagenesis represent the main obstacles to a wider\nclinical use of these vectors. Efficient and safe non-viral vectors are emerging as a promising alternative and facilitate\nclinical gene therapy studies. Here, we present an updated review for beginners and expert readers on retro and lentiviruses\nand the latest generation of transposon vectors (sleeping beauty and piggyBac) used in stable gene transfer\nand gene therapy clinical trials. We discuss the potential advantages and disadvantages of these systems such as\ncellular responses (immunogenicity or genome modification of the target cell) following exogenous DNA integration.\nAdditionally, we discuss potential implications of these genome modification tools in gene therapy and other basic\nand applied science contexts....
Long non-coding transcripts from telomeres, called telomeric repeat-containing RNA\n(TERRA), were identified as blocking telomerase activity (TA), a telomere maintenance mechanism\n(TMM), in tumors. We expressed recombinant TERRA transcripts in tumor cell lines with TA\nand with alternative lengthening of telomeres (ALT) to study effects on TMM and cell growth.\nAdeno- and lentivirus constructs (AV and LV) were established for transient and stable expression of\napproximately 130 units of telomere hexanucleotide repeats under control of cytomegalovirus (CMV)\nand human RNase P RNA H1 (hH1) promoters with and without polyadenylation, respectively.\nSix human tumor cell lines either using telomerase or ALT were infected and analyzed for TA levels.\nPre-infection cells using telomerase had 1%ââ?¬â??3% of the TERRA expression levels of ALT cells. AV and\nLV expression of recombinant TERRA in telomerase positive cells showed a 1.3ââ?¬â??2.6 fold increase in\nTERRA levels, and a decrease in TA of 25%ââ?¬â??58%. Dominant-negative or small hairpin RNA (shRNA)\nviral expression against human telomerase reverse transcriptase (hTERT) results in senescence, not\ninduced by TERRA expression. Population doubling time, cell viability and TL (telomere length)\nwere not impacted by ectopic TERRA expression. Clonal growth was reduced by TERRA expression\nin TA but not ALT cell lines. ALT cells were not affected by treatments applied. Established cell\nmodels and tools may be used to better understand the role of TERRA in the cell, especially for\ntargeting telomerase....
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