0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Resident's Forum |

In Vitro and Ex Vivo Delivery of Short Hairpin RNAs for Control of Hepatitis C Viral Transcript Expression

Bonnie E. Lonze, MD, PhD; Horatio T. Holzer, BS; Matthew K. Knabel, BS; Jayme E. Locke, MD, MPH; Gregory A. DiCamillo, BS; Sunil S. Karhadkar, MD; Robert A. Montgomery, MD, DPhil; Zhaoli Sun, MD, PhD; Daniel S. Warren, PhD; Andrew M. Cameron, MD, PhD
Arch Surg. 2012;147(4):384-387. doi:10.1001/archsurg.2011.1250.
Text Size: A A A
Published online

Recurrent hepatitis C virus (HCV) infection is the most common cause of graft loss and patient death after transplantation for HCV cirrhosis. Transplant surgeons have access to uninfected explanted livers before transplantation and an opportunity to deliver RNA interference-based protective gene therapy to uninfected grafts. Conserved HCV sequences were used to design short interfering RNAs and test their ability to knockdown HCV transcript expression in an in vitro model, both by transfection and when delivered via an adeno-associated viral vector. In a rodent model of liver transplantation, portal venous perfusion of explanted grafts with an adeno-associated viral vector before transplantation produced detectable short hairpin RNA transcript expression after transplantation. The ability to deliver anti-HCV short hairpin RNAs to uninfected livers before transplantation and subsequent exposure to HCV offers hope for the possibility of preventing the currently inevitable subsequent infection of liver grafts with HCV.

Figures in this Article

Sign in

Create a free personal account to sign up for alerts, share articles, and more.

Purchase Options

• Buy this article
• Subscribe to the journal

Figures

Place holder to copy figure label and caption
Graphic Jump Location

Figure 1. Small interfering RNA (siRNA)–mediated hepatitis C virus (HCV) transcript knockdown in vitro. Clone B cells were transfected with lacZ (control), 5′ untranslated region (272), and NS4B (NS4) siRNAs. RNA was harvested in 3 days. Quantitative polymerase chain reaction for the HCV replicon transcript was performed, and transcript expression in NS4 (light gray bars) and 272 (dark gray bars) relative to lacZ control–transfected cells (black bars) was calculated. Columns represent the mean relative expression from a minimum of 3 independent experiments per time point. Error bars indicate SEM.

Place holder to copy figure label and caption
Graphic Jump Location

Figure 2. Hepatitis B virus (HBV) and NS4B (NS4) short hairpin RNA (shRNA) expression in vitro. HEK293 cells were transfected with either pAAV-eGFP-HBV or pAAV-eGFP-NS4 plasmids as indicated, and RNA was harvested in 3 days. MicroRNA (miRNA) Northern blotting was performed and the membrane was probed with HBV-specific (A), NS4-specific (B), and miR16-specific (C) radiolabeled probes.

Place holder to copy figure label and caption
Graphic Jump Location

Figure 3. Short hairpin RNA (shRNA)–mediated hepatitis C virus (HCV) transcript knockdown in vitro. Clone B cells were transfected with either pAAV-eGFP-HBV or pAAV-eGFP-NS4 plasmids as indicated, and RNA was harvested in 3 days. Bright field (A), fluorescence (B), and merged (C) microscopic images are shown from a representative field 3 days after transfection to illustrate typical transfection efficiency. D, Northern blotting of total RNA was performed and the membrane was examined with an NS5B-specific probe for detection of the clone B replicon transcript. 18S RNA in the ethidium bromide stained gel is shown for loading control. E, The NS5B probe detects a transcript specific to clone B cells that is not expressed in the Huh-7 parental cell line. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase. F, Quantitative polymerase chain reaction was performed after reverse transcription of total RNA. The graph shows HCV transcript expression in pAAV-eGFP-NS4–transfected cells (gray bar) relative to pAAV-eGFP-HBV–transfected cells (black bar) at 3 days and represents the mean expression of 3 independent experiments. Error bars indicate SEM.

Tables

References

Correspondence

CME
Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
Submit a Comment

Multimedia

Some tools below are only available to our subscribers or users with an online account.

Sign in

Create a free personal account to sign up for alerts, share articles, and more.

Purchase Options

• Buy this article
• Subscribe to the journal

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Collections
PubMed Articles
HIV and liver transplantation: The British Columbia experience, 2004 to 2013. Can J Infect Dis Med Microbiol 2014;25(3):159-62.
Current status of kidney transplantation in HIV-infected patients. Curr Opin Nephrol Hypertens 2014;23(6):619-24.
Jobs
brightcove.createExperiences();