Handling Gene Therapy in Clinical Settings

In recombinant viral vectors, nonessential viral genes can be replaced by the therapeutic gene of interest and modified to be replication incompetent1,2

As part of the delivery vector, there is a viral capsid that can trigger various components of our immune system2,3

Because gene therapies utilise viral vectors, they are to be considered biohazardous materials and must be handled using proper procedures4,5


Defining Risk and Biosafety of Gene Therapy and Infectious Agents

To minimise the potential risk of exposure to infectious agents, basic biological risk and containment criteria have been established for their handling6–8

  • Infectious agents are classified into risk groups that describe the relative level of risk of infection6
  • The risk group to which an infectious agent is assigned is one factor used to determine the appropriate biosafety level — a set of procedures and laboratory containment for the handling of the agent8,9

The risk group classifications have been established by the NIH Guidelines and WHO and are based on the principal hazardous characteristics of the agent and the threat it poses on the individual and the community7

NIH, National Institutes of Health; WHO, World Health Organization.

Risk Group 1

Agents that are not associated with disease in healthy adult humans10

Represent no or little individual and community risk7

Risk Group 1

Examples: Bacillus subtilis; AAV (all serotypes); recombinant or synthetic AAV constructs*10

*Constructs in which the transgene does not encode either a potentially tumourigenic gene product or a toxin molecule, and are produced in the absence of a helper virus.
AAV, adeno-associated virus.

Risk Group 2

Agents that are associated with human disease that is rarely serious and for which preventive or therapeutic interventions are often available10

Represent moderate individual risk but low community risk7

Risk Group 2

Salmonella; Staphylococcus aureus; Penicillium marneffei; Toxoplasma; all human adenoviruses; hepatitis A, B, C, D, and E viruses; herpes zoster; all human papilloma viruses10

Risk Group 3

Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available10

Risk Group 3

Examples: Coxiella burnetii; Rickettsia akari; West Nile virus; yellow fever virus; SARS-CoV; monkeypox virus; transmissible spongiform encephalopathies agents; HIV types 1 and 210

Except for Phase II, the Nine Mile strain is listed as Risk Group 2.
HIV, human immunodeficiency virus; SARS, severe acute respiratory syndrome; SARS-CoV, SARS-associated coronavirus.

Risk Group 4

Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available10

High individual and community risk7

Risk Group 4

Lassa virus; herpes B or monkey B virus; Ebola virus; undefined haemorrhagic fever agents and viruses10

Biosafety Levels

Biosafety is the application of safety precautions that reduce the risk of exposure in the laboratory to a potentially infectious microbe and limit contamination of the work environment11

There are four biosafety levels designated by the CDC that include BSL-1, BSL-2, BSL-3, and BSL-47,11,12

Each level has specific containment controls for laboratory practices, safety equipment and facility construction11


BSL, biosafety level; CDC, Centers for Disease Control and Prevention.


Biosafety Level 4

Criteria for inclusion7

Dangerous/exotic agents* that pose individual risk of aerosol-transmitted infections; agents that have a close antigenic relationship to a BSL-4 agent; agents that have unknown risk of transmission

Laboratory practices7

BSL-3 practices plus:

Decontamination of all material on exit from facility

Entrance through change room and shower on exit

Safety equipment7

BSL-3 equipment plus:

Class III BSCs or

Partial containment equipment with full-body, air-supplied,
positive pressure suit


Separate building or isolated zone

Dedicated supply and exhaust, vacuum and decontamination systems

*Frequently fatal and for which there are not vaccines or treatments.
BSC, biological safety cabinets; BSL, biosafety level.


Biosafety Level 3

Criteria for inclusion7

Indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposure

Laboratory practices7

BSL-2 practices plus:

Controlled access to laboratory

Decontamination of all waste and laboratory clothing

Safety equipment7

BSL-2 equipment plus:

PPE includes protective laboratory clothing, gloves, face, eye and respiratory protection

BSC or other physical containment devices for all open manipulation of agents


Self-closing, double-door access; sealed windows and penetrations

Entry through airlock or anteroom

Hand-washing sink near exit

Exhausted air not recirculated

Available autoclave

BSC, biological safety cabinets; BSL, biosafety level; PPE, personal protective equipment.


Biosafety Level 2

Criteria for inclusion7

Agents of moderate hazard associated with human disease that can be transmitted via percutaneous injury, ingestion or mucous membrane exposure

Laboratory practices7

BSL-1 practices plus:

Limited access to laboratory

Biohazard warning signs

Safety equipment7

BSL-1 equipment plus:

PPE includes laboratory coat, gloves, face and eye protection

BSC for procedures that may cause exposure to aerosol or splashes


Self-closing doors

Sink for hand washing

Readily available eye-wash station

Available autoclave

BSC, biological safety cabinets; BSL, biosafety level; PPE, personal protective equipment.


Biosafety Level 1

Criteria for inclusion7

Agents not known to consistently cause disease in healthy adults

Laboratory practices7

Standard microbiological practices

Safety equipment7

No primary barriers required

PPE includes laboratory coat and gloves


Doors for access control

Benches or tables able to support loads and use, and easy to clean

Sink for hand washing

PPE, personal protective equipment.

Risk Groups and Biosafety Levels for Gene Therapy Vectors

Human gene transfer vectors are, at a minimal, categorised into Risk Group 1 and Risk Group 28

  • Most modern vectors are incapable of or restricted in their replication in vivo and thus considered to be nonpathogenic Risk Group 1 agents, which supports their use under BSL-1 procedures7,8
  • Despite this classification, many institutional biosafety committees require adherence to BSL-2 containment and procedures due to the unknown effects of accidental exposure and potential for generation of replication-competent vectors8
  • BSL-2 procedures are designed to prevent physical contact with cultures, offer staff prophylactic measures including vaccinations and establish methods for treating individuals who have been exposed to the agent8
  • Biosafety level classifications are inconsistent across Europe; it is important to ensure that all practices comply with the national legislation before initiation of treatment with a gene therapy12

BSL, biosafety level.

Risk Groups for Common Viral Vectors

Adeno-associated viral vector 1 1
Avipoxvirus 1 1
Modified vaccinia virus 1 1
Adenoviral vector 2 1
Herpes viral vector (HSV-1) 2 1
Lentiviral vector 3 2
Retrovirus vector 3 (MoMLV; for animals) 2 (for animals); ≤2 (for human)

*Based on the Belgian classification system; Not taking into account the transgene.
MoMLV, Moloney murine leukemia virus; WT, wild-type.

Risk Identification for Gene Therapy Products

Risk identification tools may be useful in determining appropriate safe handling procedures14

  • Given the heterogeneity of gene therapy products with regards to their integrating and hazardous properties, it is recommended that each gene therapy undergoes assessment of risk based on specific viral vector and transgene properties14
  • For gene therapy products that have the capability to replicate, integrate into the genome or contain a toxic transgene, more detailed risk evaluations are recommended by an institutional committee to determine safe handling procedures14

Decision Tree for Handling of Gene Therapy Products14


*BSL classification was recommended, provided that the gene therapy product was manufactured without using any adenovirus or any other helper virus of human origin.
BSL, biosafety level; rDNA, recombinant DNA.
Figure from Figure 1, Petrich J, et al. J Pharm Pract 2020; 33:846–855.

Examples of Potential Opportunities of Exposure in Clinical Settings

Unintended exposure to hazardous agents can occur via dermal and mucosal absorption, inhalation, injection and ingestion14

Activity Potential opportunity of exposure
Receipt Contacting residues on drug containers, individual dosage units or work surfaces
Compounding and other manipulations Constituting, reconstituting powdered, lyophilised agent
Expelling air or agent from syringes
Contacting residue present on PPE or other garments
Decontaminating work surfaces
Generating aerosols during administration of agent
Priming an IV administration set
Patient care activities
Handling bodily fluids or body fluid-contaminated materials
Spills Generation, management and disposal of spills
Transport Moving agent within a healthcare setting
Waste Collection and disposal of hazardous and contaminated waste

IV, intravenous; PPE, personal protective equipment.

Biosafety Procedures for Hazardous Drugs in Clinical Setting

All personnel handling hazardous drugs must be appropriately trained before handling the agent15


Must be stored to prevent spillage or breakage

Refrigerated agents must be stored in a dedicated refrigerator


Must be administered safely using protective medical devices and techniques

Appropriate PPE must be worn


Equipment (including tubing and needles) and packaging materials must be disposed appropriately, after administration

All areas and equipment should be thoroughly decontaminated, deactivated and cleaned

Spill Control15

In the event of a spill, the spill must be contained and cleaned immediately only by qualified personnel with appropriate PPE

Spill kits containing all essential materials must be readily available

PPE, personal protective equipment.

Biosafety Procedures for Gene Therapy

Each clinical laboratory using human gene transfer vectors must be properly equipped with the following8:

Personal protective equipment

  • Gloves
  • Eye protection
  • Face (surgical) masks
  • Lab coats

Laboratory facilities

  • Sink, eye-wash station, drench shower
  • Fume hood
  • Biosafety cabinets
  • Sharps disposal
  • Access to an autoclave
  • Doors marked with biohazard signs

Guidance on Handling of Gene Therapy Products

Although gene therapies may be considered biohazardous materials, they can be administered with minimal risk if proper procedures are followed5

Receipt and Storage5

  • All gene therapy shipments must be properly inspected and opened in the pharmacy preparation room wearing appropriate PPE
  • Must be stored at a suitable temperature, according to product information
  • Gene therapy storage areas should be labeled to alert employees of possible hazard


  • Preparer should always wear chemotherapy gloves (or two pairs of surgical gloves) and a closed front surgical-type gown with knit cuffs
  • All dose preparations must be done in a designated Class II* laminar flow biological safety cabinet

Cleaning and Disposal5

  • The biosafety cabinet must be disinfected before and after completing all gene therapy drug preparations
  • Protective clothing must always be worn
  • All disposable material should be placed in a gene therapy-specific biohazard container

*Class II hoods are partial barrier systems that rely on the movement of air to provide personnel, environmental and product/preparation protection.15
PPE, personal protective equipment.

Appropriate Use of Personal Protective Equipment for Handling Gene Therapies

Within a pharmaceutical environment, the potential routes of exposure must be considered when evaluating the necessary PPE16

  • Potential routes of exposure include accidental skin puncture, ingestion, direct contact with the skin or mucous membrane, or inhalation from an aerosol16
  • For the handling of biological products, gowns, head covers, masks, shoe covers and gloves are required16
  • Goggles should be considered when splashes or sprays are anticipated16
  • When compounding Risk Group ≥3 agents, a respirator is required16

PPE, personal protective equipment.

Summary/Module Recap

  • Basic biological risk and containment criteria have been established for the handling of infectious agents to minimise the potential risk of exposure to them7,8
  • Infectious agents are classified into risk groups that determine the appropriate biosafety level in which the agent is to be handled6,8,9
  • Medical institutions should ensure that they have plans in place to allow for the safe handling of infectious agents with minimal chance of exposure and mechanisms of quick response if such exposure occurs8


  1. Bouard D, et al. Br J Pharmacol 2009; 157: 153-165.
  2. Naso MF, et al. BioDrugs 2017; 31: 317-334.
  3. Maurer AC, et al. Cell Reports 2018; 23: 1817-1830.
  4. Pharmacy Purchasing and Products. What Safe Handling Practices Are Necessary for Gene Therapies? Available at: https://www.pppmag.com/article/1721/July_2015/What_Safe_Handling_Practices_Are_Necessary_for_Gene_Therapies/. Accessed 4 June 2020.
  5. Armitstead JA, et al. Hosp Pharm 2001; 36: 56-66.
  6. Eur-Lex. Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work. Available from: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02000L0054-20200624. Accessed 4 June 2021.
  7. U.S. Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories. 5th edn. December 2009. Available at: https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2009-P.PDF. Accessed 2 April 2019.
  8. Swindle S. Curr Protoc Hum Genet 2018; 96: 2.1.1-12.1.17.
  9. Public Health Emergency. Risk Groups. Available at: https://www.phe.gov/s3/BioriskManagement/biosafety/Pages/Risk-Groups.aspx. Accessed 3 April 2019.
  10. National Institutes of Health. NIH Guidelines. April 2019. Available at: https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.pdf. Accessed 30 July 2021.
  11. Centers for Disease Control and Prevention. Recognizing the Biosafety Levels. Available at: https://www.cdc.gov/training/QuickLearns/biosafety/. Accessed 2 April 2019.
  12. Vulto AG, et al. EJHP Pract 2007; 13: 29-39.
  13. Baldo A, et al. Curr Gene Ther 2013; 13: 385-394.
  14. Petrich J, et al. J Pharm Pract 2020; 33: 846-855.
  15. U.S. Pharmacopeia. General Chapter 800. Hazardous Drugs – Handling in Healthcare Settings. Available at: http://www.usp.org/sites/default/files/usp/document/our-work/healthcare-quality-safety/general-chapter-800.pdf. Accessed 4 April 2019.
  16. Blind JE, et al. Am J Health Syst Pharm 2019; 76: 795-80