PRODUCT SAFETY

SMARTvector™ Inducible
shRNA Lentiviral particles—
Safety information
Introduction
Safety is a focus of all lentiviral vector technology. For this reason, lentiviralvector packaging systems have divided the essential functions amongst
multiple plasmids to reduce the risk of generating replication-competent
lentivirus (RCL). The split-genome packaging system is designed so thatmultiple recombination events between the components are required forautonomous replication. Clinical trials using a split-genome packaging
system have shown that this strategy effectively eliminates the creation ofRCLs (see Levine et. al. PNAS, 103:17372-17377, 2006). Commercially available
3rd generation lentiviral vector systems separate the viral envelope, env(e.g. VSV-G) from the gag-pro-pol, which encodes structural and enzymaticfunctions. SMARTvector Inducible Lentiviral shRNA 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) from gag-pro. Because the RT and IN
enzymes are provided in trans to gag-pro, additional recombination events
are necessary to produce RCLs.

SMARTvector inducible lentiviral shRNA
lentiviral vector technology
The SMARTvector Inducible Lentiviral shRNA transfer vector consistsof a deleted HIV-1 pNL4-3 provirus that retains only those cis-acting
sequences necessary for packaging, reverse transcription and integration(Figure 1). SMARTvector Inducible shRNA lentiviral particles are 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.
mCMV
PGK
hEF1α
mEF1α

TRE3G
5' LTRΨ

ortRFP

1. A 5' long terminal repeat (LTR);
2. A primer binding site (PBS) complementary to tRNALys3 for initiation ofreverse transcription and minus strand 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 DNA
synthesis; and7. 7. A 3’ LTR that lacks the enhancer elements located in the U3 region(ΔU3) needed for native transcriptional activity from the viral LTR. Thedeletion within the 3’-LTR results in a vector that is self-inactivating (SIN).

Trans-lentiviral packaging system
The Trans-Lentiviral Packaging System (Dharmacon Cat# TLP5913) offers asuperior safety profile compared to other commercially available lentiviral
vector systems. The packaging components are separated onto fiveplasmids (Figure 2). Additionally, expression of gag-pro and tat-rev areunder the control of the conditional tetracycline-responsive promoter
element (TRE), limiting expression of these viral components strictly to thepackaging cell line. A detailed description of the Trans-lentiviral packaging
system can be found in Wu, et. al. Mol. Ther. 2(1) 47–55, 2000.

SMARTchoicepromoters

PuroR 2A Tet-On 3G WPRE 3' SIN LTR

tGFP

RRE

The viral sequences contained in the SMARTvector Inducible shRNA
lentivector genome include:

SMARTvector
universal scaffold

Figure 1. SMARTvector Inducible shRNA genome

dharmacon.horizondiscovery.com

P TRE

gag-propA

LTR HIV2

vpr-RT-IN (pol)

pA

P CMV

env (VSV-G)

P CMV

P TRE

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

General containment considerations

Tet-OffTM

tatIRES

pA

pA

revpA

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 openreading 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-INfusion 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 early
promoter. 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.

The major risks associated with the use of lentiviral vectors are (1) the
potential for generating a replication competent lentivirus; and (2) the
potential 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 andproduction of vector-based lentivirus as set by your institution’s biosafetycommitee.
• 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: oba.od.nih.gov/oba/rac/Guidance/LentiVirus_Containment/pdf/Lenti_Containment_Guidance.pdfThe Trans-Lentiviral Packaging System used to create SMARTvector Inducible
Lentiviral shRNA lentiviral particles ensures that the enduser receives a safe
product that can be effectively used in a BSL-2 environment to silence gene
expression in a wide range of cell types. It is vital that the protocols suppliedwith the viral particle product for appropriate handling and storage are fully
understood and followed precisely. We strongly advise that users of thisproduct have extensive experience with cell culture techniques.
Any investigator who purchases SMARTvector Inducible lentiviralparticle products is responsible for consulting with their institution’s
health and biosafety personnel for specific guidelines on the handling oflentiviral vector particles. Further, each investigator is fully responsiblefor obtaining the required permissions for the acceptance of lentiviralparticles into their local 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), September 2009, downloadable here:
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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