Linking the Expression of Therapeutic Genes to Unfolded Protein Response: A New Option for Anti-Hepatitis B Virus Gene Therapy.

Tight control of transgene expression is key to ensure the efficacy of a wide range of gene therapy interventions, in which the magnitude and duration of gene expression have to be adjusted to therapeutic needs, thereby limiting secondary effects. The development of upgraded strategies to link transgene expression to pathological stress episodes is an unmet need in gene therapy. Here, we propose an expression strategy that associates transgene expression to an intracellular stress coping mechanism, the unfolded protein response. Specifically, we harnessed the cis elements required to sustain the noncanonical splicing of X-box binding protein 1 (XBP1) messenger RNA (mRNA) in response to the dysfunction of the endoplasmic reticulum (ER), a situation commonly known as ER stress, to drive the expression of heterologous genes. Since ER stress features a wide variety of pathological conditions, including viral infections, cancer, or metabolic disorders, this new expression module stimulates the synthesis of therapeutic genes as a response to cellular damage, and ensures their expression only when necessary. Validation of this inducible expression system was performed in vitro and in vivo, and its potential to limit/inhibit viral infections has been shown in proof-of principle experiments.

X-box Binding Protein 1 Regulates Unfolded Protein, Acute-Phase, and DNA Damage Responses During Regeneration of Mouse Liver.

BACKGROUND & AIMS: Liver regeneration after partial hepatectomy (PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 (XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. METHODS: We performed studies with C57Bl6-J (control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. RESULTS: Expression of XBP1 by hepatocytes increased immediately after PH (priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response (DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene (Stat3). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X (H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. CONCLUSION: In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.

Safety and liver transduction efficacy of rAAV5-cohPBGD in nonhuman primates: a potential therapy for acute intermittent porphyria.

Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.

[Suprapubic cystostomy catheter knotting. Presentation of the first national case and review of the literature].

OBJECTIVE: This study presents the first Spanish case of a spontaneous knot in the catheter of a suprapubic cystostomy and review the national and international literature. METHODS: The case of an 87-year-old patient who was diagnosed with adenocarcinoma of the prostate is presented. A suprapubic vesical puncture for urinary retention was urgently performed in this patient because of the impossibility of urethral catheterisation. A spontaneous knot in the catheter was detected upon removal; the tightened knot could be removed by gentle and sustained traction without surgery. RESULTS: Knotting or calcification of the catheter was suspected when the catheter remained anchored in the bladder during a removal attempt 5 days after initial catheterisation. Plain pelvis x-ray was taken, but no calcification or knots were observed because the catheter was radiolucent. An ultrasound would have offered more information, but it was not requested. Gentle and sustained traction of the catheter reduced the knot size and allowed catheter removal without complications. Worldwide cases and national publications were reviewed. CONCLUSION: The formation of spontaneous or manipulation-induced knots in urinary cystostomy catheters is an extremely rare complication. The presented case is the first Spanish case of catheter knotting; it is only the 17th reported case worldwide.