PRODUCT SAFETYGIPZ™ shRNA lentiviral particles
safety information

Introduction

The viral sequences contained in the GIPZ shRNA lentivector genome include:

Safety is a focus of all lentiviral vector technology. For this reason,
lentiviral vector packaging systems have divided the essentialfunctions amongst multiple plasmids to reduce the risk of generating
replication-competent lentivirus (RCL). The split-genome packaging
system is designed so that multiple recombination events between
the components are required for autonomous replication. Clinical trials
using a split-genome packaging system have shown that this strategy
effectively eliminates the creation of RCLs (see Levine et. al. PNAS,
103:17372-17377, 2006). Commercially available 3rd generation lentiviralvector systems separate the viral envelope, env (e.g. VSV-G) from the gagpro-pol, which encodes structural and enzymatic functions. GIPZ shRNA
Lentiviral Particles are produced using the Trans-Lentiviral Packaging
System. The Trans-Lentiviral Packaging system provides an even higher
level of safety over 3rd generation packaging systems by further splitting
the viral pol (reverse transcriptase (RT) and integrase (IN) functions) fromgag-pro. Because the RT and IN enzymes are provided in trans to gag-pro,
additional recombination events are necessary to produce RCLs.

1. A 5' long terminal repeat (LTR);
2. A primer binding site (PBS) complementary to tRNALys3 for initiation ofreverse transcription and minusstrand DNA synthesis;
3. A sequence encompassing the HIV-1 psi (Ψ) packaging signal including a
5' terminal fragment of the gag sequence;
4. A env fragment that includes the Rev response element (RRE);
5. A 156 base pol fragment (FLAP) from coordinates 4327 to 4483 of HIV-1
SG3 molecular clone (see Ghosh, et. al. Virology, 194(2), 858-864, 1993)
containing a central polypurine tract (cPPT) and central termination site(CTS), which is required for efficient translocation of the pre-integration
complex across the nuclear envelope;
6. The polypurine track (ppt), which facilitates initiation of plus-strand DNAsynthesis; and7. A 3' LTR that lacks the enhancer elements located in the U3 region (ΔU3)
needed for native transcriptional activity from the viral LTR. The deletion
within the 3'-LTR results in a vector that is self-inactivating (SIN).

GIPZ shRNA lentiviral vector technology

Trans-lentiviral packaging system

The GIPZ shRNA transfer vector consists of a deleted HIV-1 pNL4-3 provirusthat retains only those cis-acting sequences necessary for packaging,reverse transcription and integration (Figure 1). GIPZ shRNA is replication
incompetent, containing less than 30% of the wild-type HIV-1 genome.
Furthermore, all essential replication genes, gag, pol, and env, andaccessory genes, tat, rev, vif, vpr, vpu, and nef, have been removed.

The Dharmacon™ Trans-Lentiviral Packaging System offers a superior safety
profile compared to other commercially available lentiviral vector systems.
The packaging components are separated onto five plasmids (Figure 2).
Additionally, expression of gag-pro and tat-rev are under the control of
the conditional tetracycline-responsive promoter element (TRE), limiting
expression of these viral components strictly to the packaging cell line. A
detailed description of the Trans-lentiviral packaging system can be foundin Wu, et. al. Mol. Ther. 2(1) 47–55, 2000.

∆U3
5’LTRPBSΨ∆gagRRE

FLAP

ZeoR

CMV

tGFP

IRES

puro

WPRE

ppt

3'-LTR
SIN

Figure 1. GIPZ shRNA genome

dharmacon.horizondiscovery.com

miR-30 flanking sequences

P TRE

gag-propA

LTR HIV2

vpr-RT-IN (pol)

pA

P CMV

env (VSV-G)

pA

P CMV

P TRE

Tet-OffTM

tat

IRES

pA

rev

pA

Figure 2. Trans-lentiviral packaging system

The five plasmid components for the Trans-lentiviral packaging system are
as follows:
1. pTLA1-PAKThe gag-pro packaging construct contains a DNA fragment from theHIV-1 molecular clone SG3 (coordinates 258 to 8384), which has been
modified by a 39-base deletion of the packaging signal (Ψ) and a 1357base deletion in the env gene (coordinates 5827 to 7184). The pol open
reading frames (ORFs) for the RT and IN genes were deleted by replacing
the first codon of each with stop codons (TAA). A frame-shift mutation
was introduced into the Vpr-encoding sequence by blunt-end ligation
at the SalI restriction site. Together these modifications delete theexpression of RT, IN, as well as the accessory proteins, Vpr, Vif, Vpu, andNef. Expression of Gag-Pro is controlled by a TRE promoter.
2. pTLA1-ENZ
The RT and IN proteins are packaged in the viral particle via an in-frame
fusion with the virion-associated protein Vpr. Expression of the Vpr-RT-IN
fusion polyprotein is under the control of the HIV-2 LTR, which is transactivated by the Tat protein. Description of this construct can be foundin Wu, et. al. EMBO J. 16, 5113-5122, 1997.
3. pTLA1-ENVThe VSV-G expression vector contains the VSV-G cDNA expressed froma human cytomegalovirus (CMV) immediate early promoter. In addition,
an SV40 polyadenylation signal has been cloned downstream of theVSVg cDNA.
4. pTLA1-TOFF
The Tet-Off™ expression vector contains the tetracycline-controlled
transactivator (tTA) under the control of the hCMV immediate earlypromoter. Tet-Off™ is required for transcriptional activation of Gag-Pro
expression (pTLA1-PAK) and Tat/Rev expression (pTLA1-Tat/Rev).
5. pTLA1-Tat/Rev
The HIV-1 auxiliary proteins Tat and Rev are expressed from a bicistronic
vector containing an Internal Ribosomal Entry Site (IRES). Its expressionis under control of the TRE promoter. Tat expression is required fortransactivation of the 5' LTRs found in GIPZ shRNA and pTLA1-ENZ viralvectors. Rev is required for nuclear export and translation of mRNAtranscripts encoding the gag-pro and vpr-RT-IN (pol) genes, as well as
the full-length viral genome.

In addition, each of the packaging plasmids contains a pUC origin ofreplication for propagation in E. coli. and the beta lactamase (bla) gene for
ampicillin resistance.

General containment considerations
The major risks associated with the use of lentiviral vectors are (1) thepotential for generating a replication competent lentivirus; and (2) thepotential for oncogenesis due to insertional activation/inactivation of key
regulatory genes; for example, activation of an oncogene or inactivation ofa tumor suppressor.
To mitigate these risks, please follow the safety guidelines for use
and production of vector-based lentivirus as set by your institution’s
biosafety commitee.
• For glycerol stocks of E. coli. containing lentiviral plasmids, BSL1
guidelines should be followed• For handling and use of lentiviral products to produce lentiviral particles,
BSL2 or BSL2+ guidelines should be followed• For handling and use of lentiviral particle products, BSL2 or BSL2+guidelines should be followed
For guidance on containment for lentiviral vectors, please refer to theRecombinant DNA Advisory Committee (RAC) guidelines for researchwith lentiviral vectors: https://osp.od.nih.gov/wp-content/uploads/Lenti_
Containment_Guidance.pdf
The Trans-lentiviral packaging system used to create GIPZ shRNA Lentiviral
Particles ensures that the enduser receives a safe product that can beeffectively used in a BSL-2 environment to silence gene expression in awide range of cell types. It is vital that the protocols supplied with the viralparticle product for appropriate handling and storage are fully understoodand followed precisely. We strongly advise that users of this product haveextensive experience with cell culture techniques.
Any investigator who purchases GIPZ lentiviral particle products isresponsible for consulting with their institution’s health and biosafety
personell for specific guidelines on the handling of lentiviral vector
particles. Further, each investigator is fully responsible for obtaining therequired permissions for the acceptance of lentiviral particles into theirlocal geography and institution.
In the US, download the U.S. Department of Health and Human Services
Centers for Disease Control and Prevention and National Institutes ofHealth, Biosafety in Microbiological and Biomedical Laboratories (BMBL),
Fifth Edition, Feb 2007 here: cdc.gov/biosafety/publications/index.htm
See also: NIH Guidelines For Research Involving Recombinant DNA
Molecules (NIH Guidelines), downloadable here:
https://osp.od.nih.gov/biotechnology/nih-guidelines

t +44 (0) 1223 976 000 (UK) or +1 800 235 9880 (USA); +1 303 604 9499 (USA)f + 44 (0)1223 655 581
w horizondiscovery.com/contact-us or dharmacon.horizondiscovery.com/service-and-support
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Tet-Off is a trademark of Tet Systems. All trademarks are the property of Horizon Discovery Company unless otherwise specified. ©2018 Horizon
Discovery Group Company—All rights reserved. First published August 2014. UK Registered Head Office: Building 8100, Cambridge Research Park,Cambridge, CB25 9TL, United Kingdom.

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