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Center for Molecular Medicine

Leuven Viral Vector Core

 

Picture of Vector and Molecular Imaging GroupThis research topic historically spun out of our HIV research and links the laboratory of molecular virology (Z. Debyser) with the laboratory of neurobiology (V. Baekelandt).

Viral vector core : production and development of HIV-based lentivectors, MLV-based retrovectors and AAV-based vectors

Our viral vector technology-platform is primarily based on HIV-derived lentiviral vector technology (LV), a spin-off from our HIV research on the one hand (in parallel we also produce MLV-based viral vectors), and on AAV-based vectors The platform is supported by basic scientific research on the virology of HIV.

Lentiviral vector technology

Vectors for gene transfer derived from lentiviruses have been developed in the late nineties. Lentivirinae are a family of complex retroviruses with the human immunodeficiency virus type 1 (HIV-1) as the prototypical virus. Contrary to the oncovirinae, lentivirinae can replicate in non-dividing cells. Lentiviral vectors are produced by triple transient transfection of a packaging plasmid encoding viral structural and replicative proteins, a transfer plasmid that contains all cis-acting regulatory sequences and the transgene of interest driven by an internal promoter, and an envelope encoding plasmid (Figure 2). Lentivectors are pseudotyped with the glycoprotein of the Vesicular Stomatitis Virus (VSV-G), resulting in a wide tropism and increased stability during concentration.

Production of lentiviral vectors by triple transient transfection

 

 

 

 

 

 

 

 

 

 

 

 

 

Viral vector core : production and development of HIV-based lentivectors and AAV-based vectors

HIV-derived lentivector technology

During recent years we have expanded our collection of lentiviral vector plasmids. Our portfolio of transfer plasmids enables a broad range of applications:

  • Standard vectors : universal promoters – exchangeable cassettes – multicistronic constructs – tagged vectors
  • Imaging Vectors : eGFP – Firefly luciferase (fLuc) – Renilla luciferase (rLuc) – LacZ – HSV truncated thymidine kinase (HSV-tTK) for bioluminescence and PET imaging.
  • Regulatable vectors : -vector systems using KRAB-regulated Tetracyclin/Doxycyclin ON/OFF system (knock-down and overexpression) or FlexSwitch technology for use in Cre transgenic mice
  • RNA interference vectors for stable knock-down : shRNA mU6 and hH1 based RNAi-vectors (polIII promoters) - miRNA-based RNAi vectors (polII promoters)
  • Alternative pseudotyping : VSV-G, Rabies G, Mokola, Ebola (NTDL-6), Ampho MLV, LCMV

 

Currently, we are producing lentiviral vectors for several groups in Flanders and abroad in scientific collaborations or on a fee-for-service basis. As a partner in the KUL Imaging facility, we produce Imaging vectors and/or stable cell lines for imaging.
We put considerable effort in upscaling, fine-tuning and standardizing lentiviral vector production. The proprietary lentiviral production method is based on the production of lentiviral vectors in serum-free conditions, thereby limiting the systemic immune response in animal models. In order to carry out large-scale and reproducible animal experiments, we scaled-up our production method and improved the recovery of the lentivector from the cell supernatant by combination of tangential flow filtration (TFF) and ultracentrifugation (UCF).
At present lentivectors are routinely produced in 10-layer cell-factories (10-CF) and concentrated by TFF and UCF. The quality of each lentiviral vector production is assessed by p24 ELISA (pg p24/ml), and by estimation of transducing units (TU/ml) by limiting dilution titration of 293T cells. RNA copies are determined by Q-RT-PCR. All vectors are guaranteed to be mycoplasma and endotoxin free.

 

AAV-vector technology

Adeno-associated viral (AAV) vectors have a great potential for the treatment of both acquired and inherited disease through gene therapy. Even though the advantages of the technology for in vivo gene therapy are clear, several critical problems have surfaced that hinder its successful progression to the clinic. The efficient and selective transduction of the therapeutic target and the prevention and/or control of an immunological response to the viral gene therapy, remain important challenges to the field.
AAV-vector production is performed according to an established protocol. Following triple transient transfection in 293T cells AAV-vectors are produced. We use a plasmid encoding the transgene cassette flanked by AAV2 ITRs, a packaging plasmid coding for the rep gene of AAV2 and the cap gene of the requested serotype (e.g. AAV7), resulting in AAV2/7 viral vectors. A third plasmid provides adenoviral helper functions. Currently, we can produce AAV vectors with AAV1-AAV2-AAV7 and AAV9 serotypes.

We have optimized in-house several production and purification/concentration methods for different serotypes. DNase-resistant particles are ultimately titered using Q-PCR to assess a physical titer for each production. AAV-vectors are produced under mycoplasma- and endotoxin-free conditions. Our conditions allow a routine production of high-titer AAV-vector stocks (>5x1012 particles/mL). We are expanding the collection of AAV vectors to provide comparable transfer plasmids as in the LV portfolio, inasmuch the limited packaging capacity of AAV (<4.5 kb) allows this.

CONTACT: Dr. Rik Gijsbers; rik.gijsbers@med.kuleuven.be
For imaging vectors: Dr. Annelies Michiels; annelies.michiels@med.kuleuven.be