Developing use for recombinant lentiviral vectors for delivery of anti-hepatitis B virus micro RNA mimics

Persistent infection with hepatitis B virus (HBV) is associated with an increased risk for development of serious life-threatening complications such as cirrhosis and hepatocellular carcinoma (HCC). This common malignancy is aggressive and has a very poor prognosis. Currently available HBV therapies have variable efficacy and do not diminish the risk for development of HCC associated with infection with the virus. Consequently the development of novel therapies aimed at providing effective treatment for chronic HBV infection remains a medical priority. The therapeutic potential of harnessing the RNA interference (RNAi) pathway to achieve specific and potent silencing of pathology-causing genes has been explored for the development of novel and improved therapy to counter chronic HBV infection. Highly effective expressed anti-HBV RNAi-based sequences have been demonstrated to be capable of mediating impressive silencing of viral gene expression in vitro and in vivo. Nevertheless, accomplishing stable delivery to hepatocytes and sustained, long-term expression of therapeutic effecters remains the greatest challenge impeding the clinical translation of RNAi-based anti-HBV gene therapy. Stable integration of transgenes that may be achieved with recombinant lentiviruses, derived from the human immunodeficiency virus type 1 (HIV-1), makes these vectors particularly useful for attaining sustained expression of RNAi activators. This property is suited to countering HBV persistence, making these delivery vehicles advantageous for gene transfer of expressed RNAi anti-HBV sequences to infected hepatocytes. To advance potential clinical application of RNAi-based anti-HBV gene therapy, second generation self-inactivating (SIN) lentiviral vectors were engineered to include liver specific, RNA polymerase II (Pol II) cassettes that generate HBV-silencing primary micro RNA (pri-miR) mimics. The antiviral pri-miR sequences, placed under the control of the liver-specific mTTR promoter, are derived from natural pri-miR-31 and engineered to generate anti-HBV RNAi guide sequences targeting single (monocistronic pri-miR sequences) or multiple (polycistronic pri-miR sequences) sites within the highly conserved multifunctional HBV X protein (HBx) open reading frame (ORF) of the viral genome. The HBV silencing lentiviruses (LVs) stably transduced liver-derived Huh7 and HepG2.2.15 cells. Northern blot analysis of RNA extracted from stably transduced Huh7 cells verified that the integrated anti-HBV pri-miR sequences were processed to form RNAi-activating guide strands according to the intended design. When stably transduced Huh7 cells were transfected with a HBV replication-competent plasmid, potent inhibition of markers of viral replication was achieved. Sustained, hepatotropic HBV silencing was effected with limited disruption of endogenous miR function. Silencing of a mutated HBV sequence in stably transduced cells was caused by HBV-silencing LV expressing a polycistronic pri-miR effecter. Rigorous evaluation of the anti-HBV efficacy of the polycistronic silencing LV in the HepG2.2.15 HBV replication cell line demonstrated potent antiviral activity. Preclinical analysis of the efficacy of the RNAi-activating lentiviral vector system was assessed using the HBV transgenic mouse model. HBV silencing LVs encoding a liver-specific polycistronic pri-miR 31/5-8-9 sequence was delivered to young adult animals via a tail-vein injection as well as to neonatal mice via a superficial temporal vein injection. The HBV-silencing LV was capable of mediating stable hepatic delivery and sustained expression of anti-HBV pri-miR sequences in vivo. Furthermore, the therapeutic sequences were processed in vivo according to the intended design and mediated powerful knockdown of HBsAg, circulating viral particle equivalents and intrahepatic viral RNA without inducing hepatotoxicity. To our knowledge this is the first study to evaluate the efficacy of RNAi-activating anti-HBV lentiviral vectors in the HBV transgenic mouse model. Although the investigation constitutes a preliminary step towards clinical application of HBV silencing RNAi-based lentiviral vectors, this study has provided proof of principle for a therapeutic strategy that can be potentially useful for the treatment of chronic HBV infection

Evaluation of peripapillary choroidal and retinal nerve fiber layer thickness in eyes with tilted optic disc

Purpose: This study was performed to evaluate the retinal nerve fiber layer (RNFL) and peripapillary choroidal thickness in eyes with tilted optic disc in order to identify characteristic RNFL and peripapillary choroid patterns verified by optical coherence tomography (OCT). Methods: Twenty-nine eyes of 29 patients with tilted optic discs were studied with spectral-domain (SD)-OCT and compared with age and sex-matched control subjects in a prospective design. The imaging of RNFL was performed using circular scans of a diameter of 3.4 mm around the optic disc using OCT. For measurements of peripapillary choroidal thickness, the standar d protocol for RNFL assessment was performed. Results: SD-OCT indicated significantly lower superotemporal (p<0.001), superonasal (p=0.001), and global (p=0.005) RNFL thicknesses in the tilted disc group than those of the control group. Peripapillary choroid was significantly thicker at the site of the elevated rim of eyes with tilted disc (p<0.001). Conclusion: This study demonstrated a clinical characterization of the main tilted disc morphologies that may be helpful in differentiating a tilted disc from other altered disc morphologies. Further studies are recommended to study the comparison between glaucoma and tilted disc groups

Assessment of efficacy of miRNA-regulated TetR-KRAB control of transgene expression in vivo.

<p>(<b>a</b>) Schematic diagram of the AAV vectors used for in vivo studies. (<b>b</b>) GFP fluorescence was measured in live animals at 8 weeks after injection with recombinant AAVs. Mice received AAVs encoding the TetR-KRAB sequence without copies of miRNA target located downstream. Animals did or did not receive doxycycline in their drinking water. The color scale next to the images indicates the signal intensity. (<b>c</b>) <b>&</b> (<b>d</b>) Results at 8, 12 and 16 weeks post injection are expressed in counts/mm²/second. Data shown are mean and error bars indicate the SD. * Statistically significant differences <i>P</i><0.05 (two-tailed, unpaired Student’s <i>t</i>-test), n = 4 per group.</p

Lentiviral vectors used to target transgene expression to skeletal muscle-derived cells.

<p>(<b>a</b>) Schematic diagram of the lentivirus vectors used to accomplish skeletal muscle-specific expression. (<b>b</b>) miR-133 expression levels in C2C12d, C2C12ud and 293T cells detected by RT-qPCR. Three independent experiments were performed in duplicate. *Statistically significant differences <i>P</i> = 0.0185 (two-tailed, unpaired Student’s <i>t</i>-test). (<b>c</b>) Differentiated or undifferentiated C2C12 cells (C2C12d and C2C12ud respectively) were transduced with lentiviral vectors carrying the TetR-KRAB sequence followed by four copies of miRNA target of miR-122 or miR-133. After 10 days of culture with or without doxycycline, transduced cells were analyzed for GFP expression by FACS. Results are expressed as a percent of MFI. The raw data corresponding to 100% are indicated above each histogram bar. Data shown are mean and error bars indicate the SD of 3 independent experiments performed in triplicate.</p

miRNA-based regulation of TetR-KRAB control of transgene expression.

<p>(<b>a</b>) Schematic representation of the miRNA-TetR-KRAB regulatory and reporter cassettes. (<b>b</b>) In the absence of tissue-specific miRNA, the repressor is translated and is free to bind the tetO operator, thus preventing transgene expression. However in the presence of a tetracyclin analog (Doxycycline) the repressor does not bind and transcription is activated.(<b>c</b>) In targeted cells tissue-specific miRNA binds to its complementary target and results in degradation of the TetR-KRAB mRNA. As a result, transgene expression occurs.</p

miR-122 concentrations and reporter gene expression in transduced cells.

<p>(<b>a</b>) miR-122 expression levels in Huh7 and 293T cells detected by RT-qPCR. Three independent experiments were performed in duplicate. *Statistically significant differences <i>P</i> = 0.03 (two-tailed, unpaired Student’s <i>t</i>-test). (<b>b</b>) <b>&</b> (<b>c</b>) GFP expression in transduced Huh7 and 293T cells. After 10 days of culture, with or without doxycycline, transduced 293T (<b>b</b>) or Huh7 (<b>c</b>) cells were analyzed for GFP expression by FACS. Results are expressed as a percentage of MFI. In the TetR-KRAB system, the expression of GFP is optimal in the presence of doxycycline and corresponds to 100% of the MFI. The raw data corresponding to 100% are indicated above each histogram bar. (<b>d</b>) RT-qPCR of GFP mRNA normalized to values obtained for 18S RNA. Data shown are mean and error bars indicate the standard deviation (SD) of 3 independent experiments performed in triplicate. (<b>e</b>) Representative images of GFP expression in cells at 10 days after transduction. Lentiviral vectors encoding TetR-KRAB mRNA with four target sequences for miR-122 and GFP gene under the control of a liver specific promoter in 293T and Huh7 cells with or without doxycycline treatment. Magnifications ×50. (<b>f</b>) Representative high power fields showing immunofluorescence staining of TetR-KRAB (red) in Huh7 cells transduced with recombinant lentiviruses. Four target sequences for miR-122 were absent (left panel) or present (right panel) in the TetR-KRAB-expressing cassettes of the vectors, which also produced GFP (green) constitutively. Magnifications ×400.</p

Lentiviral vectors used to target transgene expression to macrophage-derived cells.

<p>(<b>a</b>) Schematic diagram of the lentivirus vectors used to target cells of immune lineage. (<b>b</b>) miR-142 expression levels in Huh7, 293T and NR8383 cells detected by RT-qPCR. Three independent experiments were performed in duplicate. * Statistically significant differences <i>P</i>  = 0.043 (two-tailed, unpaired Student’s <i>t</i>-test). (<b>c</b>) <b>&</b> (<b>d</b>) Huh7, 293T and NR8383 cells were transduced with two different lentiviral vectors carrying the TetR-KRAB sequence follow by 4 copies of miR-142 target (c) or miR-122 (d). After 10 days of culture with or without doxycycline, transduced cells were analyzed for GFP expression by FACS. Results are expressed as a percentage of mean fluorescence intensity (MFI). In TetR-Krab system, the expression of GFP is optimal in the presence of doxycycline, corresponding to 100% of the MFI. The raw data corresponding to 100% are indicated above each histogram bar.Data shown are mean and error bars indicate the SD of 3 independent experiments performed in triplicate.</p

Schematic diagram of the recombinant lentivirus vectors used to accomplish liver-specific transgene expression.

<p>(<b>a</b>) <b>&</b> (<b>b</b>) Lentiviral vector encoding four target sequences of miR-122, TetR-KRAB and GFP under the control of a mTTR liver specific promoter (<b>a</b>) or constitutively active CMV promoter (<b>b</b>). (<b>c</b>) Lentiviral vector without target sequences of miR-122 and expressing GFP from liver-specific mTTR promoter. (<b>d</b>). Inducible TetR-KRAB lentiviral vector system that includes a GFP gene under control of a CAG promoter as part of a bicistronic unit comprising the KRAB based repressor (PLVCT). cPPT, central polypurine tract; IRES, internal ribosomal entry site; WPRE, woodchuck hepatitis virus post-transcriptional element; RRE: Rev protein responsive element; pA, polyadenylation site; CMV, cytomegalovirus; CAG, CMV immediate enhancer/β-actin; PGK, phosphoglycerate kinase; mTTR,murine liver-specific transthyretin receptor promoter.</p