Working with Cell-Penetrating Peptide (CPP) Trans-Activating Tranductions (TATs):

Risk Assessment and Biosafety Recommendation

Gema Puspa Sari

Budiman BelaVirology and Cancer Pathobiology Research Center for

Health Service (VCPRC) FKUI-RSCM

The 2nd Asian Conference on Safety and Education in Laboratory

Introduction

• Cell-Penetrating Peptide (CPP)– Able to translocate across the plasma membrane of

eukaryotic cell– Use for intracellular delivery

• Trans-Activating Tranductions (TATs):– Potent trans-activator of HIV-1– Essential for viral replication– Able to trans-activate of other cellular genes– Can leave cells from which is synthesized and cross

the membrane of adjacent cell, where it localizes in the nucleus

The 2nd Asian Conference on Safety and Education in Laboratory

• Amino acid sequence of HIV-1 TAT

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg

• Potential hazardous of TAT protein:– Immunosuppressive -> induce apoptosis CD4+ T cell,

induce abnormalities in macrophage and APC

– Cytotoxic activity-> expression of HIV-1 TAT on brain can cause a number of developmental and behavioral abnormalities

– Carcinogenic property -> stimulation of cell growth and has potent angiogenic activity

The 2nd Asian Conference on Safety and Education in Laboratory

Risk Assessment

• TAT transduction domains only:– not infectious

– not replicate

– not transfer any genetical material

– Sero-conversion due to TAT-residues are very scarce

• Nature of the proteins being fused to TAT possibly:– Immunogenic

– Toxins

– Oncogen

• Fusion proteins potential:– Enter through skin

– Induce MHC-I

The 2nd Asian Conference on Safety and Education in Laboratory

Biosafety Recommendation (1)

• Biosafety Level 1:

– Cloning of sequences coding for TAT-fusion proteins in E. coli

– Plasmid preparation

The 2nd Asian Conference on Safety and Education in Laboratory

Biosafety Recommendation (2)

• Biosafety Level 2: Expression of TAT-fusion protein

– Proteins must be handled as hazardous material

• Labcoat with front close, mouth pippetting is not allowed, masker, disposable gloves, safety google, avoid direct contact with the skin, cuts, or mucous membrane

• Work in BSC or plastic backed absorbent lab paper in bench

The 2nd Asian Conference on Safety and Education in Laboratory

Biosafety Recommendation (3)

• Decontamination Procedure

– Wear PPE (labcoat, gloves, goggle, masker)

– Decontaminate work surface using detergent with a protease enzyme for minimum 20 minutes

The 2nd Asian Conference on Safety and Education in Laboratory

Disposal Procedures

• Deactivate and dispose of TAT solutions and cultures using standard autoclave methods

• Deactivate solution using a 1:10 dilution of bleach (sodium hypoclorite) in a 1:1 mixture with TAT solution

– Dispose of solution down the sewer drain with copius amounts of water

The 2nd Asian Conference on Safety and Education in Laboratory

Wash hand after working with TAT material

The 2nd Asian Conference on Safety and Education in Laboratory

References

Becker-Hapak M, McAllister SS, Dowdy SF. “TAT-mediated protein transduction into mammalian cells”. Methods. 2001 Jul; 24(3):247-56. Review.

Backus, B.D., Dowdy, S.F., Boschert, K.R., and Richards, T.L, Becker-Hapak, M. (2000). “Safety Guidance for Laboratory Personnel Working with Trans-Activating Transduction (TAT) Protein Transduction Domains”. American Chemical Society, Journal of Chemical Health and Safety. 2001 March/April.

General position statement of the ZKBS on the risk assessment of the expression of Tat-fusion proteins, May 2006 http://www.bvl.bund.de/SharedDocs/Downloads/06_Gentechnik/ZKBS/02_Allgemeine_Stellungnahmen_englisch/08_vectors/zkbs_vectors_Tat_fusion_proteins_2006.pdf?__blob=publicationFile&v=1

Kim BO, Liu Y, Ruan R, et al. Neuropathologies in Transgenic Mice Expressing Human Immunodeficiency Virus Type 1 Tat Protein under the Regulation of the Astrocyte-Specific Glial Fibrillary Acidic Protein Promoter and Doxycycline. American Journal of Pathology, Vol. 162, No. 5, May 2003.

Schwarze SR, Hruska KA, Dowdy SF. “Protein transduction: unrestricted delivery into all cells?” Trends Cell Biol. 2000 Jul; 10(7):290-5. Review.

Swiss Expert Committee for Biosafety. Risk Assessment of Work with TAT- or VP22- Fusion Proteins. www.efbs.admin.ch

University of Kentucky. Department of Biological Safety. Minimum Guidelines for TAT-Fusion Protein Use. http://ehs.uky.edu/biosafety/

Watson K, Edwards RJ. HIV-1 trans-Activating (Tat) Protein: Both a target and a tool in therapeutic approaches. Biochem Pharmacol 58;10:1521–1528, 1999.

Working with Cell-Penetrating Peptide (CPP) Trans-Activating Tranductions (TATs):

Risk Assessment and Biosafety Recommendation

Gema Puspa Sari, Budiman BelaVirology and Cancer Pathobiology Research Center for Health Service (VCPRC) FKUI-RSCM

Corresponding author: gemapuspasari@yahoo.com

AbstractCell-penetrating peptides (CPPs) are peptides which able to cross cellular membrane and able to be a non-invasive vector1 since it can bring other molecules such as small RNA/ DNA, plasmids, antibodies, and nanoparticles to be transported into cells as its cargo.2 Trans-activating transduction (TAT) proteins are 14 kDa proteins transcribed from complex spliced mRNAs which function intracellularly as a trans-activating factor of the human immunodeficiency virus type 1 (HIV-1).3 Some studies showed that exposure to Tat protein can lead to a serious health consequences even in the absence of HIV infection4 because it has potential immunosuppressive, cytotoxic, and carcinogenic properties.

Keywords: Cell-penetrating peptides (CPP), Trans-activating transduction (TAT) protein, Human immunodeficiency virus type 1 (HIV-1), health consequences

References1. Trabulo S, Cardoso AL, Mano M, et al. 2010. Cell-penetrating peptides – mechanisms of cellular uptake and

generation of delivery systems. Pharmaceuticals 3; 961-993.2. de Figueiredo IR, Freire JM, Flores L, et al. Cell-penetrating peptides: a tool for effective delivery in gene-targeted

therapies. IUBMB 66 (3); 182-194.3. Rusnati M, Taraboletti G, Urbinati C, et al.2000. Thrombospondin-1/HIV-1 Tat protein interaction: modulation of the

biological activity of extracellular Tat. The FASEB Journal 14:1917-1930.4. Kim Bo, Liu Y, Ruan Y, et al. 2003. Neuropathologies in transgenic mice expressing human immunodeficiency virus

type 1 Tat protein under the regulation of the astrocyte-specific glial fibrillary acidic protein promoter and doxycycline. Am J Pathol 162 (5): 1693-707.