Gene Therapy Progress and Prospects: Recent progress in transgene and RNAi expression cassettes

Ill, Charles R.; Chiou, Henry C.

doi:10.1038/sj.gt.3302524

Cited by 22 publications

(14 citation statements)

References 41 publications

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“…[22][23][24] Thus, to avoid methylation-induced shut off of transgenes in vivo, the current credo strives to remove CpGs from both, the vector backbone and the expression cassette. 11,25,26 Contrary to popular belief, we have recently reported that the depletion of CpG dinucleotides from a green fluorescent protein (GFP) gene results in a significant reduction of in vitro expression in transiently and stably transfected cells suggesting a methylationindependent effect of intragenic CpGs on gene expression. 27 In the present study, we have evaluated the influence of rational gene design on the expression level, longevity and bioactivity of the model gene murine erythropoietin (mEPO) in electroporated mice.…”

Section: Introductionmentioning

confidence: 79%

Minimal doses of a sequence-optimized transgene mediate high-level and long-term EPO expression in vivo: challenging CpG-free gene design

Kosovac

Wild

Ludwig³

et al. 2010

Gene Ther

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Advanced gene delivery techniques can be combined with rational gene design to further improve the efficiency of plasmid DNA (pDNA)-mediated transgene expression in vivo. Herein, we analyzed the influence of intragenic sequence modifications on transgene expression in vitro and in vivo using murine erythropoietin (mEPO) as a transgene model. A single electro-gene transfer of an RNA-and codon-optimized mEPOopt gene into skeletal muscle resulted in a 3-to 4-fold increase of mEPO production sustained for 41 year and triggered a significant increase in hematocrit and hemoglobin without causing adverse effects. mEPO expression and hematologic levels were significantly lower when using comparable amounts of the wild type (mEPOwt) gene and only marginal effects were induced by mEPODCpG lacking intragenic CpG dinucleotides, even at high pDNA amounts. Corresponding with these observations, in vitro analysis of transfected cells revealed a 2-to 3-fold increased (mEPOopt) and 50% decreased (mEPODCpG) erythropoietin expression compared with mEPOwt, respectively. RNA analyses demonstrated that the specific design of the transgene sequence influenced expression levels by modulating transcriptional activity and nuclear plus cytoplasmic RNA amounts rather than translation. In sum, whereas CpG depletion negatively interferes with efficient expression in postmitotic tissues, mEPOopt doses o0.5 mg were sufficient to trigger optimal long-term hematologic effects encouraging the use of sequence-optimized transgenes to further reduce effective pDNA amounts.

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Section: Introductionmentioning

confidence: 79%

Minimal doses of a sequence-optimized transgene mediate high-level and long-term EPO expression in vivo: challenging CpG-free gene design

Kosovac

Wild

Ludwig³

et al. 2010

Gene Ther

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“…This technique is based on the fact that, despite multiple trinucleotide sequences encoding for a single amino acid, certain codons are preferred over others depending on the host organism (mostly due to tRNA frequencies) (209). By making silent mutations within the transgene, it is thus possible to increase translation efficiency by optimizing codon usage for the target cell; this strategy also allows for other changes, such as the removal of negative regulatory cis -acting features, to enhance expression (210). Codon optimization of F.IX was reported to increase expression 3-4-fold compared with the unaltered sequence (63).…”

Section: Gene Therapies For Hemophlia Bmentioning

confidence: 99%

Gene therapy for hemophilia

Rogers

Herzog

2015

Front Biosci

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Hemophilia is an X-linked inherited bleeding disorder consisting of two classifications, hemophilia A and hemophilia B, depending on the underlying mutation. Although the disease is currently treatable with intravenous delivery of replacement recombinant clotting factor, this approach represents a significant cost both monetarily and in terms of quality of life. Gene therapy is an attractive alternative approach to the treatment of hemophilia that would ideally provide life-long correction of clotting activity with a single injection. In this review, we will discuss the multitude of approaches that have been explored for the treatment of both hemophilia A and B, including both in vivo and ex vivo approaches with viral and nonviral delivery vectors.

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“…In microRNA (miRNA) biogenesis, many miRNAs are processed from long pol II transcripts (Bartel 2004;Ill and Chiou, 2005;Kim, 2005;Lee et al, 2004) called primary microRNAs (pri-miRNAs). These transcripts, often several kb in length, consist of imperfect double-stranded stems separated by single-stranded sequences (Lee et al, 2002).…”

mentioning

confidence: 99%

RNA Interference from Multimeric shRNAs Generated by Rolling Circle Transcription

2006

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Methods most commonly used for producing small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) are chemical synthesis and intracellular expression from engineered vectors. For shRNAs, chemical synthesis is very costly and construction of vectors is laborious. Synthesis by phage RNA polymerases from their natural promoters results in a 5 -terminal triphosphate that can trigger an interferon (IFN) response. Moreover, due to the requirement of phage promoters for 5 - GPuPuPu sequences for transcription initiation, shRNA transcripts may have extra 5 -nucleotides that can constrain the sequences that can be targeted. Also, the 3 ends may have an additional n + 1 nucleotide not encoded by the template. Here we present a novel approach for synthesizing functional shRNAs via rolling circle transcription (RCT) of small (approximately 70 nt) single-stranded DNA circles using T7 RNA polymerase, which avoids these issues. Due to internal pairing, these circles are dumbbell-shaped. RCT produces large transcripts (>10 kb in length) consisting of multimers (>150 copies) of shRNAs in the absence of promoter, terminator, or primer sequences. Dumbbells targeting red fluorescent protein (DsRed), human tumor necrosis factor-alpha (TNF-alpha) and hepatitis C virus (HCV) internal ribosome entry site (IRES) were prepared and transcribed. The resulting long transcripts are substrates for Dicer. When introduced into 293FT and Huh7 cells, the multimeric transcripts inhibited their target genes at levels similar to an equivalent mass of monomeric shRNAs, indicating that they can enter the RNAi pathway. Thus, rolling circle transcription of small DNA dumbbells provides a new source of biologically active interfering RNA.

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Gene Therapy Progress and Prospects: Recent progress in transgene and RNAi expression cassettes

Cited by 22 publications

References 41 publications

Minimal doses of a sequence-optimized transgene mediate high-level and long-term EPO expression in vivo: challenging CpG-free gene design

Minimal doses of a sequence-optimized transgene mediate high-level and long-term EPO expression in vivo: challenging CpG-free gene design

Gene therapy for hemophilia

RNA Interference from Multimeric shRNAs Generated by Rolling Circle Transcription

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