Ex Vivo Gene Delivery to Hepatocytes: Techniques, Challenges, and Underlying Mechanisms

The neogenesis of insulin-producing cells (IPCs) from non-beta-cells has emerged as a potential method for treating diabetes mellitus (DM). Many groups have documented that activation of pancreatic transcription factor(s) in hepatocytes can improve the hyperglycemia in diabetic mice. In the present study, we explored a novel protocol that reprogrammed primary hepatocytes into functional IPCs by using multicistronic vectors carrying pancreatic and duodenal homeobox-1 (Pdx1), neurogenin 3 (Ngn3), and v-musculoaponeurotic fibrosarcoma oncogene homolog A (MafA). These triple-transfected cells activated multiple beta-cell genes, synthesized and stored considerable amounts of insulin, and released the hormone in a glucose-regulated manner in vitro. Furthermore, when transplanted into streptozotocin-induced diabetic mice, the cells markedly ameliorated glucose tolerance. Our results indicated that ectopic expression of Pdx1, Ngn3, and MafA facilitated hepatocytes-to-IPCs reprogramming. This approach may offer opportunities for treatment of DM.

show abstract

“…Our data indicated that transfection efficiency was 29.3 ± 1.1% (Figure 1), which was close to previous study [14]. …”

Section: Resultssupporting

confidence: 92%

Reprogramming of Mice Primary Hepatocytes into Insulin-Producing Cells by Transfection with Multicistronic Vectors

Luo

Chen

Liu

et al. 2014

Journal of Diabetes Research

View full text Add to dashboard Cite

show abstract

“…The first was to use a lentivirus expressing a myc-tagged SRSF3. We were able to produce this lentivirus in HEK-293T cells, but primary rat hepatocytes are exceptionally resistant to lentiviral infection ([38, 43] and data not shown). Another potential alternative we considered was to use an adeno-associated vector (AAV), but AAV systems also require splice-site switching [44] and thus we would likely encounter the same issues.…”

Section: Resultsmentioning

confidence: 99%

“…Hepatocytes in primary culture recapitulate liver-specific traits better than cell lines or hepatomas; however, disadvantages to their use include their poor transfection efficiency and relatively short lifespan in culture (< 5 days) [38]. Adenoviral infection provides a mechanism to express proteins in > 95% of hepatocytes in as little as 24 h after infection, and can also be used in intact animals for liver-specific gene expression.…”

Section: Introductionmentioning

confidence: 99%

Construction and evaluation of an adenoviral vector for the liver-specific expression of the serine/arginine-rich splicing factor, SRSF3

Suchanek

Salati

2015

Plasmid

View full text Add to dashboard Cite

Serine/arginine-rich splicing factor-3 (SRSF3), alternatively known as SRp20, is a member of the highly-conserved SR protein family of mRNA splicing factors. SRSF3 generally functions as an enhancer of mRNA splicing by binding to transcripts in a sequence-specific manner to both recruit and stabilize the binding of spliceosomal components to the mRNA. In liver, expression of SRSF3 is relatively low and its activity is increased in response to insulin and feeding a high carbohydrate diet. We sought to over-express SRSF3 in primary rat hepatocytes to identify regulatory targets. A standard adenoviral shuttle vector system containing an epitope-tagged SRSF3 under the transcriptional control of the CMV promoter could not be used to produce infectious adenoviral particles. SRSF3 over-expression in the packaging cell line prevented the production of infectious adenovirus particles by interfering with the viral splicing program. To circumvent this issue, SRSF3 expression from the shuttle vector was blocked by placing its expression under the control of the liver-specific albumin promoter. In this system, the FLAG-SRSF3 transgene is only expressed in the target cells (hepatocytes) but not in the packaging cell line. An additional benefit of the albumin promoter is that expression of the transgene does not require the addition of hormones or antibiotics to drive SRSF3 expression in the hepatocytes. Robust expression of FLAG-SRSF3 protein is detected in both HepG2 cells and primary rat hepatocytes infected with adenovirus prepared from this new shuttle vector. Furthermore, abundances of several known and suspected mRNA targets of SRSF3 action are increased in response to over-expression using this virus. This report details the construction of the albumin promoter-driven adenoviral shuttle vector, termed pmAlbAd5-FLAG.SRSF3, that can be used to generate functional adenovirus to express FLAG-SRSF3 specifically in liver. This vector would be suitable for over-expression of other splicing factors that could inhibit virus production. In addition, this vector would allow only liver-specific expression of other cargo genes when used in a whole-animal paradigm.

show abstract

“…For example, the ex vivo culture of primary hepatocytes is challenging, the hepatocytes may not survive the genetic manipulation, and the engraftment of manipulated hepatocytes back in in vivo is also not optimal. 93 Another aspect to consider is the potential toxicity associated with the gene targeting. First of all, the different nuclease platforms may be seen by the immune system as foreign, as shown in vivo in mice.…”

Section: Key Pointmentioning

confidence: 99%

Therapeutic editing of hepatocyte genome in vivo

Lujambio

2017

Journal of Hepatology

View full text Add to dashboard Cite

Ex Vivo Gene Delivery to Hepatocytes: Techniques, Challenges, and Underlying Mechanisms

Cited by 13 publications

References 43 publications

Reprogramming of Mice Primary Hepatocytes into Insulin-Producing Cells by Transfection with Multicistronic Vectors

Reprogramming of Mice Primary Hepatocytes into Insulin-Producing Cells by Transfection with Multicistronic Vectors

Construction and evaluation of an adenoviral vector for the liver-specific expression of the serine/arginine-rich splicing factor, SRSF3

Therapeutic editing of hepatocyte genome in vivo

Contact Info

Product

Resources

About