Gene therapy is a technique that modifies a person's genes to treat or cure disease. Gene therapies can work by several mechanisms: Replacing a disease-causing gene with a healthy copy of the gene. Inactivating a disease-causing gene that is not functioning properly. Genetic therapies hold promise to treat many diseases, but they are still new approaches to treatment and may have risks. Potential risks could include certain types of cancer, allergic reactions, or damage to organs or tissues if an injection is involved. Recent advances have made genetic therapies much safer. Gene therapy is on course to revolutionize medical care for several conditions. The hope is that gene therapy will be a one-time curative therapeutic intervention for diseases ranging from inherited hemoglobinopathies, such as sickle cell disease and thalassemia, to acquired diseases such as HIV.

1. Biosafety Considerations
MEDWEB—US-00124

2. Criteria used
to categorize
biological risk
The history and
current state of
human gene therapy
Risk management
when working with
adeno-associated
virus (AAV) vectors
Criteria used to
ensure appropriate
containment
procedures

4. ADA-SCID, severe combined immunodeficiency due to adenosine deaminase deficiency; CAR, chimeric antigen receptor.
Timeline adapted from: Wirth T, et al. Gene 2013;525:162–169.
1. Rosenberg SA, et al. N Engl J Med 1990;323(9):570–578. 2. Blaese RM, et al. Science 1995;270(5235):475–480. 3. Sibbald B. CMAJ 2001;164(11):1612.
4. BioPharm International 2008;17(5). http://www.biopharminternational.com/genesis-gendicine-story-behind-first-gene-therapy.
5. Wirth T, et al. Gene 2013;525:162–169. 6. Hoggatt J. Cell 2016;166(2):263. 7. US FDA. Approved drugs. August 30, 2017.
https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm574154.htm. 8. US FDA. Approved drugs. October 18, 2017
https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm581296.htm. 9. FDA Release. December 19, 2017.
https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm589467.htm. All accessed June, 2018.
China first country
to approve a gene
therapy-based
product for
clinical use4
First successful
phase 3 gene
therapy clinical
trial in the EU5
Europe approves
GSK2696273 to treat
patients with ADA-
SCID6
US approves
axicabtagene
ciloleucel (CAR-T cell
therapy) for large B-
cell lymphoma8
US approves
tisagenlecleucel
(CAR-T cell therapy)
for leukemia7
US approves first
directly administered
gene therapy,
voretigene
neparvovec for
retinal dystrophy9
First therapeutic
gene transfer in
ADA patients2
The death of gene
therapy clinical trial
patient, Jesse
Gelsinger3
First officially
approved gene
transfer into
humans (Steven A.
Rosenberg)1
Retrovirus vector

5. Figures reproduced from The Journal of Gene Medicine, © 2017 John Wiley and Sons Ltd. Gene Therapy Clinical Trials Worldwide, December 2018. Available at: http://www.abedia.com/wiley/indications.php (left chart); Accessed
April 08, 2019. http://www.abedia.com/wiley/vectors.php (right chart); Accessed April 08, 2019.
Cancer 66.6% (n=1951) Monogenic diseases 11.5% (n=338)
Infectious diseases 6.3% (n=184) Cardiovascular diseases 6.2% (n=183)
Neurological diseases 1.8% (n=52) Ocular diseases 1.3% (n=37)
Inflammatory diseases 0.5% (n=15) Other diseases 2.2% (n=63)
Gene marking 1.7% (n=50) Healthy volunteers 1.9% (n=57)
Adenovirus 18.5% (n=541) Retrovirus 17.5% (n=514)
Naked/ Plasma DNA 15.4% (n=452) Adeno-associated virus 8.1% (n=238)
Lentivirus 9.5% (n=278) Vaccinia virus 4.5% (n=133)
Poxvirus 2.4% (n=71) Herpes simplex virus 3.4% (n=99)
Other vectors 14.4% (n=420) Unknown 6.3% (n=184)

6. The Journal of Gene Medicine, © 2017 John Wiley and Sons Ltd. Gene Therapy Clinical Trials Worldwide, December 2018. Available at: http://www.abedia.com/wiley/phases.php. Accessed April 08, 2019.
0
10
20
30
40
50
60
70
80
90
100
Phase 1 (n=1644) Phase 1/2 (n=634) Phase 2 (n=485) Phase 2/3 (n=29) Phase 3 (n=122) Phase 4 (n=5) Single subject (n=11)
%
of
Gene
Therapy
Clinical
Trials

7. Collins M & Thrasher A. Proc Biol Sci 2015;282(1821).
Cells are extracted from the patient, modified with the
therapeutic gene, and injected back into the patient
The therapeutic gene is transferred directly to target
cells in the body
The genetically modified
cells (e.g. stem cells) are
multiplied in the laboratory
The therapeutic
transgene is introduced
into a delivery cell such as
a stem cell that is often
derived from the patient
...and re-administered
to the patient
Therapeutic transgene
The therapeutic transgene
is packaged into a delivery
vehicle such as a virus
Target organ
(e.g. liver)
... and injected into
the patient
Therapeutic transgene
The therapeutic transgene
is packaged into a delivery
vehicle such as a virus

8. AAV, adeno-associated virus; ADA-SCID, severe combined immunodeficiency due to adenosine deaminase deficiency; EMA, European Medicines Agency;
FDA, US Food and Drug Administration; HSCT, hematopoietic stem cell transplantation; HSV-1, Herpes simplex virus type 1.
1. Imlygic Prescribing Information. 2. Kymriah Prescribing Information. 3. Yescarta Prescribing Information. 4. Luxturna Prescribing Information. 5. Imlygic SmPC.
6. Strimvelis SmPC. 7. Zalmoxis SmPC. Hyperlinks last accessed September 2018.
FDA-approved therapy Indication (approval date) Delivery Viral vector
Imlygic (talimogene
laherparepvec)1 Melanoma (2015) In vivo HSV-1
Kymriah (tisagenlecleucel)2 B-cell precursor acute lymphoblastic leukemia (2017);
diffuse large B-cell lymphoma (2018)
Ex vivo Lentivirus
Yescarta (axicabtagene
ciloleucel)3 Large B-cell lymphoma (2017) Ex vivo Retrovirus
Luxturna (voretigene
neparvovec-rzyl)4 Biallelic RPE65 mutation-associated retinal dystrophy (2017) In vivo AAV
EMA-approved therapy Indication (approval date)
Imlygic5 Melanoma (2015) In vivo HSV-1
Strimvelis6 ADA-SCID (2016) Ex vivo Retrovirus
Zalmoxis7 Adjunctive treatment in HSCT in high-risk blood cancer (2016) Ex vivo Retrovirus

9. Reproduced from Wang D, et al. Discov Med 2014;18:151–161.
Genetic factors
and/or
environmental factors
Therapeutic gene
Addition
Alleviation
Disease
Functional gene
Correction
Disease
Mutant gene
Replacement
For monogenic diseases; involves replacing a mutated
gene that causes disease with a healthy gene
For complex and infectious diseases; introduces
a new gene into the body to help fight a disease,
often to supplement a targeted therapeutic agent

10. Reproduced from Wang D, et al. Discov Med 2014;18:151–161.
Correction
Disease
Mutant gene
Gene silencer
Correction
Disease
Mutant gene
Knockdown
TTTTTTTTTTTTTT TTTTTTTTTT
TTTTTTTTTT
TTTTTTTTTT
Inactivating a mutated gene that is over-producing its
product by targeting RNA
Making a targeted change to the gene sequence
Gene corrector

11. 1. Collins M, et al. Proc R Soc B 2015;282:20143003.
2. Keeler AM, et al. Clin Transl Sci 2017;10:242–248.
3. Wang D, et al. Discov Med 2014;18:151–161.
The aim of Gene Replacement Therapy – or GRT –is to provide sufficient gene expression
in enough targeted cells to ameliorate or correct dysfunctional phenotype1
Recessive disorders1,2, single gene
mutations3, and X-linked diseases2
Dominant disorders where an errant
gene codes for destructive or
interfering proteins2
Implementation3 • Either directly in vivo or through ex vivo cell therapy
• Vehicle/vector used to deliver therapeutic transgene
✘
✓

12. Thomas CE, et al. Nat Rev Genet 2003;4(5):346–358.
Retrovirus
Lentivirus
Genomes integrate into
the host genome
Integrating
Herpes Simplex Virus type 1 (HSV-1)
Adeno-Associated Virus (AAV)
Adenovirus
Persist in the
cell nucleus
predominantly as
extrachromosomal
episomes
Generally
non-integrating
Monogenic diseases represent 11.1% of diseases studied with gene therapy, with most gene therapy
trials performed in cancers

13. GRT, gene replacement therapy.
1. Wang D, et al. Discov Med 2014;18:67–77.
2. Ayuso E, et al. Curr Gene Ther 2010;10:423–436.
• the vector, or vehicle, which is injected
into the patient and by which a transgene
is delivered to the targeted cells1
• the transgene, which is a sequence of
complementary DNA (cDNA) coding the
replacement gene1
• the promoter, which is the DNA sequence
that acts as a “turn on” switch and modulates
the expression of the transgene1
• A termination signal to end gene transcription1, or
• Inverted terminal repeats (ITRs) at either end of
the cassette to allow for synthesis of
complementary DNA2
Adapted from Wang D. Discov Med 2014;18:67–77.
Promoter Transgene
Termination
Signal

15. AAV, adeno-associated virus; GRT, gene replacement therapy.
1. Deyle DR, Russell DW. Curr Opin Mol Ther 2009;11(4):442–447. 2. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. April 2016. https://osp.od.nih.gov/wp-
content/uploads/2013/06/NIH_Guidelines.pdf. 3. Gonçalves MA, et al. Virol J 2005;2:43–60. 4. Gombash SE. Postdoc J 2015;3:1–12; Environmental Health & Safety. Adeno-Associated Virus and Adeno-Associated Viral Vectors.
https://ehs.research.uiowa.edu/adeno-associated-virus-and-adeno-associated-viral-vectors (hyperlinks last accessed September 2018).
• rAAV-based vectors used in GRT have wildtype viral genes removed and typically exist as
extrachromosomal episomes1,3
• AAV can efficiently infect both non-dividing and dividing cells1
• AAV is typically transmitted by respiratory and gastrointestinal routes1,4
AAV is a member of the parvovirus family of single-stranded small DNA viruses1
AAV
Requires a helper virus such as adenovirus or herpes simplex virus for replication1
Considered a Risk Group 1 microorganism – infection with AAV is not associated with disease in
humans2
Has several serotypes that impact tropism (susceptible tissues), but all appear to be non-
pathogenic3

16. Graphic from Akst J. The Scientist. June 2012.
Naso MF, et al. BioDrugs 2017;31:317–334.
1. Virus is taken into the cell via the endosome
2. The endosome breaks down
3. Therapeutic DNA enters cell nucleus as a double-
stranded molecule ready for transcription
4. Therapeutic DNA forms a circular episome
5. Upon promoter activation, transcription occurs
6. The resulting transcript leaves the nucleus and travels
to the ribosome for translation (protein synthesis)
5
6

17. Graphic from Ayuso E, et al. Curr Gene Ther 2010;10:423–436.
1. McArty DM, et al. Mol Ther 2008;16:1648–1656.
2. Ayuso E, et al. Curr Gene Ther 2010;10:423–436.
Purification produces
vectors with high titer, high
potency, and high purity2
QA using FDA-established
requirements and
predetermined
specifications3,4

18. AAV, adeno-associated virus.
1. Negrete A, Kotin RM. Brief Funct Genomic Proteomic 2008;7:303–311. 2. Naso FN, et al. BioDrugs 2017;31:317–334. 3. Watakabe A, et al. Neurosci Res 2015;93:145–157. 3. NIH Guidelines. https://osp.od.nih.gov/wp-
content/uploads/2013/06/NIH_Guidelines.pdf (last accessed September 2018). 4. Vandamme C et al. Hum Gene Ther 2017;28:1061–1074.
Transgenes remain transcriptionally active indefinitely, making AAV vectors efficient one-time
delivery vehicles, but transcription cannot subsequently be deactivated1
Although wildtype viral genes have been deleted from the AAV vector, the therapeutic genes
present in AAV vectors may have unexpected properties (if non-self)2,3
Prior exposure to wild-type AAV virus has the potential to result in an immune response to the
AAV capsid; however, little to no capsid-specific cellular response has been seen in immune-
privileged tissues, such as the CNS4

19. AAV, adeno-associated virus; GRT, gene replacement therapy.
1. Foust KD & Kaspar BK. In: Spinal Muscular Atrophy: Disease Mechanisms and Therapy. London, UK: Academic Press. 2017;313–323.
2. Collins M, et al. Proc R Soc B 2015;282:20143003. 3. Wang D, et al. Mol Ther Methods Clin Dev 2018;9:234–246.
• Gene replacement therapy (GRT) aims to provide sufficient gene expression in enough targeted cells
to ameliorate or correct a dysfunctional phenotype
• AAVs are one of the most promising vectors for gene transfer and expression

20. NIH, National Institutes of Health; WHO, World Health Organization.
1. NIH. NIH guidelines for research involving recombinant or synthetic nucleic acid molecules. April 2019. Available at: https://osp.od.nih.gov/wp-content/uploads/2013/06/NIH_Guidelines.pdf. Accessed November 27, 2019; 2. U.S.
Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories – 5th Edition. December 2009. Available at: https://www.cdc.gov/labs/pdf/CDC-
BiosafetyMicrobiologicalBiomedicalLaboratories-2009-P.PDF. Accessed November 27, 2019.
Agents that are not
associated with disease in
healthy adult humans
Agents that are associated
with human disease that is
rarely serious and for
which preventive or
therapeutic interventions
are often available
Agents that are associated
with serious or lethal human
disease for which preventive
or therapeutic interventions
may be available (high
individual risk but low
community risk)
Agents that are likely to
cause serious or lethal
human disease for
which preventive or
therapeutic interventions
are not usually available
(high individual risk and
high community risk)

21. NIH, National Institutes of Health; WHO, World Health Organization.
1. NIH. NIH guidelines for research involving recombinant or synthetic nucleic acid molecules. April 2019. Available at: https://osp.od.nih.gov/wp-content/uploads/2013/06/NIH_Guidelines.pdf. Accessed November 27, 2019; 2. U.S.
Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories – 5th Edition. December 2009. Available at: https://www.cdc.gov/labs/pdf/CDC-
BiosafetyMicrobiologicalBiomedicalLaboratories-2009-P.PDF. Accessed November 27, 2019.
• Standard microbiological
practices
• Open bench or table permitted
• Lab coat and gloves
• Sink for washing hands
• Means for controlling access
(e.g. door)
• Access to work area limited when
work is conducted
• PPE incudes mask and eye
protection of face shield
• BSC for procedures that may
cause exposure to aerosol or
splashes
• Access to autoclave
• Work area includes self-closing
doors and access to eye wash
station
• Receive immunization for
microbes used
• Access restricted at all times
• BSC for all open procedures
• Exhausted air cannot be
recirculated
• Two sets of self-closing locked
doors for entrance
• Immediate access to autoclave
• Dedicated lab clothing
• Shower upon exit
• Class III BSC or full-body air
supplied suit
• Separate building or isolated zone
• Dedicated air supply and
processed exhaust

22. AAV, adeno-associated virus.
CDC BMBL. https://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf (last accessed September 2018).
High-risk
biological agents
Low-risk
biological
agents
Microbes that are not known to cause disease in
healthy adults
Practices
• Standard microbiological practices
• Open bench or table permitted
• Decontaminate work surfaces; use disinfectant
following spills; decontaminate materials before
disposal using an effective method
Equipment
• Lab coat and gloves
Facilities
• Sink for washing hands
• Means for controlling access (e.g. door)

23. AAV, adeno-associated virus.
CDC BMBL. https://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf (last accessed September 2018).
High-risk
biological agents
Low-risk
biological
agents
Microbes that pose moderate risk to workers and
environment
• Example: Staphylococcus aureus
• Some facilities may choose to use BSL-2 precautions
for AAV vectors
Practices
• Access to work area limited when work is conducted
Equipment
• PPE includes mask and eye protection of face shield
• BSC for procedures that may cause exposure to
aerosol or splashes
• Access to autoclave
Work area includes self-closing doors and access to
eye wash station

24. BSC, biological safety cabinets.
CDC BMBL. https://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf (last accessed September 2018).
High-risk
biological agents
Low-risk
biological
agents
Microbes that can cause serious or potentially
lethal disease
• Example: Mycobacterium tuberculosis
(tuberculosis)
Practices
• Receive immunization for microbes used
• Access restricted at all times
Equipment
• BSC for all open procedures
Facilities
• Exhausted air cannot be recirculated
• Two sets of self-closing locked doors for
entrance
• Immediate access to autoclave

25. BSC, biological safety cabinets.
CDC BMBL. https://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf (last accessed September 2018).
High-risk
biological agents
Low-risk
biological
agents
Very few labs in the world
Most exotic and dangerous microbes
(e.g. Ebola virus)
Practices
• Dedicated lab clothing
• Shower upon exit
Equipment
• Class III BSC or full-body, air-supplied suit
Facilities
• Separate building or isolated zone
• Dedicated air supply and processed exhaust

26. AAV, adeno-associated virus; BSL, biosafety level; EPA, Environmental Protection Agency.
*In which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule, and construct is produced in the absence of a helper virus.
1. IBC Guidance on Biosafety Level Assignment for AAV. https://www.pittsburgh.va.gov/Research/docs/AAV-Guidance.pdf. 2. Gonçalves MA, et al. Virol J 2005;2:43–60. 3. Vector Safety Information.
https://gtp.med.upenn.edu/uploads/attachments/cjgo427wq00oit1gd35bjpgmi-vector-safety-information.pdf; 4.United States EPA.
Selected EPA-registered Disinfectants. https://www.epa.gov/pesticide-registration/selected-epa-registered-disinfectants (hyperlinks last accessed September 2018).
rAAV* is considered Risk Group 1 –
infection with AAV is not associated with disease in humans1
rAAV vectors are replication-incompetent (non-infectious) by design2
Absent of hazardous genes*, rAAV vectors can be handled at BSL-1 containment1,3
rAAV vectors are susceptible to common disinfectants approved for bloodborne
pathogens (e.g. EPA Lists B, D, and E)4

27. AAV, adeno-associated virus; BSC, biological safety cabinets; BSL, biosafety level.
1. NIH Guidelines. https://osp.od.nih.gov/wp-content/uploads/2013/06/NIH_Guidelines.pdf.
2. CDC BMBL. https://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf.
3. Environmental Health & Safety. Adeno-Associated Virus and Adeno-Associated Viral Vectors. https://ehs.research.uiowa.edu/adeno-associated-virus-and-adeno-associated-viral-vectors (hyperlinks last accessed September
2018).
Use good standard
microbiological
practices and BSL-1
containment1
Conduct all open
procedures with AAV
vectors inside of BSC
(class not specified
for BSL-1)1
Use of BSC at higher levels
may be used to ensure
sterility of product
Wear lab coat, gloves,
and mask/face shield
when working with
AAV vectors2
Wash hands with
soap and water after
working with AAV
vectors2
In the event of exposure:3
• Wash exposed area with soap and water
• In event of eye exposure, flush in eye wash for 15 minutes
• Report incident per institutional policy

28. AAV, adeno-associated virus.
1. Le Guiner C, et al. Methods Mol Biol 2011;807:339–359; 2. Swedish Medical Products Agency 2. Summary notification information format for the release of genetically modified organisms other than higher plants in accordance with
Article 11 of Directive, 2001/18/EC. Available at: https://lakemedelsverket.se/upload/halso-och-sjukvard/kliniska-provningar/SNIF%20201797435.pdf. Accessed November 27, 2019.
• Use disinfectants approved for bloodborne pathogens (EPA Lists B, D, E)2
• 10% dilution of household bleach is effective (freshly prepared)
• Red containers with biohazard symbol
• Yellow chemo/biohazard mixed waste containers
• Ask your hazardous waste disposal company if you are not sure
Limit access to work area by untrained staff while working with AAV vectors3

29. AAV, adeno-associated virus; BSL, biosafety level; CDC, Centers for Disease Control and Prevention; NIH, National Institutes of Health; OSHA, Occupational Safety & Health Administration; WHO, World Health Organization.
1. NIH Guidelines. https://osp.od.nih.gov/wp-content/uploads/2013/06/NIH_Guidelines.pdf. 2. CDC BMBL. https://www.cdc.gov/biosafety/publications/
bmbl5/BMBL.pdf. 3. WHO LSM. http://www.who.int/csr/resources/publications/biosafety/en/Biosafety7.pdf. 4. OSHA. Healthcare Wide Hazards. (Lack of) Universal Precautions.
https://www.osha.gov/SLTC/etools/hospital/hazards/univprec/univ.html. 5. Gonçalves MA, et al. Virol J 2005;2:43. 6. IBC Guidance on Biosafety Level Assignment for AAV. https://www.pittsburgh.va.gov/Research/docs/AAV-
Guidance.pdf. 7. Environmental Health & Safety. Adeno-Associated Virus and Adeno-Associated Viral Vectors. https://ehs.research.uiowa.edu/adeno-associated-virus-and-adeno-associated-viral-vectors (hyperlinks last accessed
September 2018).
Basic biological risk and containment criteria have been established by the NIH1, CDC2, WHO3, and
OSHA4
The biological risks from working with AAV vectors are considered very low5
AAV vectors can be handled at BSL-1, the lowest biosafety level6
Care should nevertheless be taken to mitigate exposure to AAV vectors7
All materials contaminated with AAV vectors should be disposed of as biohazardous waste7