Gene therapy and infection control

Table I.

		Recommended Transmission Precautions by Route of Administration*
Vector	Biosafety Level	Intravenous	Intramuscular or Intratumoral	Aerosol	Intradermal
Murine retroviruses	BSL-2	S	N/A	N/A	N/A
Lentiviruses	BSL-3	S	N/A	N/A	N/A
Adenovirus at </= 1013 <!––>pfu<!––>/dose	BSL-2	S	S	C, D	N/A
Adenovirus at > 1013 pfu/dose	BSL-2	C, D	C, D	A, C, D	N/A
AAV	BSL-1	S	S	C, D	N/A
Poxviruses	BSL-1 or 2¹	N/A	N/A	N/A	S
Herpesvirus	BSL-2	S	S	N/A	N/A
Plasmid DNA	BSL-1	S	S	S	N/A

General recommendations for infection control in gene therapy

In evaluating what infection control measures are required for a given vector consider:

• the likelihood of shedding of the vector or a vector that has become replication competent

• the likely mode of transmission of the vector to other persons

• the nature of populations at risk for secondary infection

• the effect of
  <!–transgene –>expression in persons with secondary infection

The nature and extent of infection control precautions should be determined for each study by infection control personnel working with members of their local Institutional Review Board (IRB) and Institutional Biosafety Committee (IBC).

Role of Infection Control

• Notify all hospital service areas that have employees who, within the scope of their work activities, may encounter gene therapy patients, products, or waste

• Educate employees within these service areas to promote safe patient and product handling to minimize employee exposure

• Track gene therapy patients admitted to the hospital and provide them with information about necessary infection control precautions

• Work with study personnel to ensure the safety of patients, healthcare workers, and others in the event a gene therapy patient requires unanticipated services or medical care within the hospital

• Assume authority to stop any study where infection control is not being performed according to the recommendations of the IBC and Infection Control Committee

• Investigate nosocomial exposures of patients, healthcare workers, or others in the hospital to infectious gene therapy vectors

• Keep the Infection Control Committee appraised of gene therapy activities

Admissions

• Patients should be treated only in areas approved by the IBC

• Depending on the vector and route of administration, it might be of value to ensure that these areas, and waiting rooms for these areas, are physically separated from areas frequented by immmunocompromised patients who are not part of the gene therapy protocol

• If possible, rooms should be private, and with a sink and commode in the room, and appropriate transmissions precautions signs posted on the door

• Depending on the vector, it may be prudent to restrict patients to their room during treatment and limit visitors. Tests and procedures requiring patient transport from the room should be postponed if possible.

• Dedicated equipment (stethoscopes, sphygmomanometers, thermometers, etc.) should be available or should be disinfected appropriately before being reused.

Pharmacy considerations

• Vector preparations shipped from a commercial vendor should be brought into the healthcare facility through the pharmacy.

• The Pharmacy should ensure safe receipt, preparation, dispensing, and storage of gene therapy products and maintain records of their use.

• The preparation of vectors should be done by pharmacists who have training in biosafety and an understanding of infection control requirements.

• It may be of value for pharmacists to consult with the vector manufacturer for recommendations on the safe handling of the material, since, in most cases, the supplier has dealt with issues of disinfection and sterility in the manufacturing process.

• Work should not be performed on an open bench, but rather in an appropriate biosafety cabinet (BSL-1, -2, -3).

• Vacuum lines should be fitted with a HEPA filter, and centrifugation should be done in closed containers with sealed rotors as appropriate for the required bisafety level.

• Hoods and work surfaces should be decontaminated after use with an EPA-registered germicidal agent (Table II).

  Table II.

  Recommended disinfectants by vector

  Vector	Sodium hypochlorite*	Iodophor	Alcohol	Quaternary ammonium compound
  Murine retrovirus	Yes
  Lentivirus	Yes
  Adenovirus	Yes			Yes
  AAV	Yes			Yes
  Poxvirus	Yes	Yes	Yes	Yes
  Herpesvirus	Yes	Yes	Yes	Yes
  Plasmid DNA	Yes

  *a 1:10 dilution of household bleach

• Areas where infectious materials are stored should be locked and labeled with a biohazard sign indicating the nature of the agent.

• Caution should be exercised in preparing live viral vectors in the same pharmacy hood as chemotherapy and other products.

• In the absence of a dedicated hood, consideration should be given to preparing gene therapy vectors in a research laboratory.

• Protocols for the safe transport of the product from the pharmacy to the patient’s bedside should be established, and protocols should be in place for preparing for the administration of the gene therapy agent at the bedside. These should include techniques to clear air from syringes or intravenous line tubing to prevent aerosols.

Disposing of gene therapy waste

• Obviously contaminated materials (i.e. glass, vials, sharps, syringes, etc.) should be placed in a puncture-resistant container with an easily recognized biohazard label. Personal protective equipment, gauze, or other soft waste should be disposed of in red biohazard bags and handled as other regulated waste.

Emergency spill procedures

• Management of spills should depend on the amount of material spilled.

• Small spills (<10mL) should be wiped up, and the surface disinfected with an appropriate EPA-registered germicidal agent (Table II).

• Kits should be prepared in advance to manage these spills and be readily available in areas where the vector will be prepared or administered.

• For larger spills, personnel should evacuate the area; notify the appropriate authorities, warn others, control traffic, dispose of contaminated clothing and materials, clean contaminated skin with soap and water, and clean up the spill using an appropriate disinfectant.

Employee Health

• Employees who will work closely with gene therapy patients, products, or waste should be informed of the risks and hazards, and be trained to minimize any risks.

• Immunocompromised employees should be discouraged from working on gene therapy protocols.

• The Principal Investigator should meet with Employee Health personnel and develop protocols for the management of exposed healthcare workers before any patient is enrolled or treated.

• The protocol should include details about the vector and transgene, known or potential risks, recommended screening tests, treatment and follow-up, the timing of follow-up, and ways to contact investigators for consultation at all times when patients are being treated in the hospital or clinics.

• Immunization, if available, should be made available to healthcare workers.

Admissions to the Emergency Department

• Infection Control should be notified immediately if a gene therapy patient is admitted to the Emergency Department.

• Patients should be placed in a private room in Droplet/Contact Precautions unless advised otherwise by Infection Control.

Elements to consider in informed consent

• Patients undergoing gene therapy may have some unique points to consider when providing informed consent.

• They should be informed about the likelihood of generating replication competent vector through
  <!–complementation –>or
  <!–recombination–> and what this may mean in terms of infection from a wild-type virus or the vector or the effects of transgene expression.

• They should also be apprised of the potential risk for transmission of the vector, or a replication-competent recombinant to the recipient’s family or other close (e.g., sexual) contacts.

• They should be made aware of the possible need for isolation procedures during clinic visits or hospitalization.

• Patients should agree to inform medical personnel in other hospitals and clinics where they seek care that they are involved in a gene therapy protocol.

Recommended disinfectants by vector

See Table II for the recommended disinfectants by vector.

Additional details about common viral vectors and non-viral vectors

<!–Adenovirus –>vectors

• Administered
  <!–in vivo.–>

• Published trials with vector doses <=/ 10¹³ pfu have not demonstrated vector shedding or significant numbers of
  <!–RCA–>s. There is little data on the safety of trials using vector doses >10¹³pfu and for that reason it may be prudent to use Droplet and Contact Precautions for volunteers in those studies. Shedding has been reviewed recently
  <!––>PUBMED:17880045<!––>.

• Because of the possibility of recombination between vector and wild-type virus and complementation of vector by wild-type virus, it may be prudent to screen patients and their health-care workers for adenoviral infections. For the same reasons patients who acquire wild-type adenoviral infections or are exposed to adenoviral infections during gene therapy should be monitored for signs of infection and for shedding. If symptoms develop, they should be placed in Droplet and Contact Precautions. Healthcare workers exposed to symptomatic patients should be observed for signs of adenoviral infection for at least two weeks and taken out of patient care activities if symptoms develop.

• Adenovirus survives on surfaces for up to one month.

• The virus is transmissible by contact with fomites, droplets, or close personal contact.

• Wild-type virus is associated with serious disease in bone marrow transplant patients (up to 60% mortality) and liver and renal transplant patients (up to 20% mortality), and no effective therapy is available.

• Engineering–there are four gene regions (E1, E2, E3, E4) expressed early in infection that encode proteins necessary for regulating subsequent gene expression needed for viral replication. Deletions or modifications to these regions are made to cripple the virus. In some cases, all viral genes except the ITRs and contiguous packaging sequences are deleted. Since these recombinants are not capable of replication, they are grown to quantity for use in gene therapy in a
  <!–packaging cell line–>. Human cell lines such as HEK 293 that carry portions of the viral genome and stably express the gene product(s) missing from the vector have been used. Occasionally, recombination between the vector and the viral gene sequences in the packaging cell line occur leading to the generation of RCAs. A new packaging cell line (PER.C6 (Crucell, N.V., Leiden, The Netherlands)) has been developed that does not have sequence overlap with adenovirus vectors. This virtually eliminates the problem of RCA generation by homologous recombination.

<!–Retrovirus –>vectors–murine leukemia virus (MLV)

• Administered
  <!–ex vivo –>usually in the research laboratory under carefully controlled conditions. This is because MLV is readily inactivated by human complement and cannot be introduced directly into the bloodstream (recently, complement resistant murine retrovirus vectors have been developed to overcome this limitation.)

• MLV does not seem to cause human disease, so vectors probably do not present a risk even if accidentally inoculated into the bloodstream. Integration of retroviral RNA into the host genome, however, occasionally results in malignant transformation.

• When engineered for gene therapy, genes such as gag, pol, or env, required for retrovirus replication are deleted, and the transgene inserted in their place. These substitutions leave the virus crippled and unable to replicate. For gene therapy, the crippled virus must be grown to sufficient quantities to infect host cells. This is done in a
  <!–packaging cell line–>.

Retrovirus vectors–lentiviruses

• Administered in vivo.

• Like MLV, lentiviral vectors are generated by co-expressing the virus packaging elements and the vector genome in a packaging cell line. In the case of HIV-1, six of the 9 viral genes are deleted leaving only gag, pol, and rev. Such extensive deletions in the original genome make it extremely unlikely that the parental virus could be reconstituted. In addition, the vectors are engineered to be replication incompetent by selecting sequences in the
  <!–LTR/ITR–>. These sequences are essential for control of gene expression. Vectors engineered in this way are referred to as SIN (self-inactivating) vectors. Lentiviruses are not inactivated by complement so they can be infused directly into the bloodstream.

<!–Adeno-Associated Virus –>(AAV) vectors

• Administered in vivo

• Infection Control recommendations for adenoviruses should be sufficient since transmission of AAV may be similar.

• No guidelines for the number of allowable replication-competent virus in a patient dose

• Does not cause a known human disease

• AAV contains two genes, rep and cap that encode polypeptides necessary for replication and encapsidation, respectively. Replication-defective recombinants can be constructed by removing all internal viral coding sequences of the wild-type virus and inserting the transgene in their place. Expression of the transgene is then under control of the ITR sequences. Gene therapy can be accomplished using either AAV plasmid
  <!––>transfection <!––>or packaged viral particle
  <!––>transduction<!––>. Efficient propagation of the vector in a gene therapy volunteer or their contacts is unlikely because it requires both superinfection by wild type AAV (to supply the normal AAV genes) and co-infection with a helper virus such as the adenovirus. AAV’s relatively small size limits the amount of DNA that can be transduced (up to 5kb).

<!––>Poxviruses<!––>

• Poxvirus vectors are designed from vaccinia strains that may be more or less attenuated or from fowl pox or canary pox which do not infect humans. In general, highly attenuated vaccinia and animal pox viruses do not create productive infections and are less of an Infection Control concern than non-highly attenuated strains.

• Vaccination can be offered to healthcare personnel who are exposed to clinical materials contaminated with non-highly attenuated vaccinia strains used to develop vaccine recombinants.

• Vaccination is not indicated for healthcare personnel who are exposed to clinical materials contaminated with highly attenuated poxvirus strains or animal poxviruses used to develop vaccine recombinants.

• Vaccinated healthcare workers may continue to work with patients, including immunocompromised patients, as long as the vaccination site is covered and good hand washing is observed.

• Poxviruses persist in the environment for only 6 to 24 h depending upon ambient temperature and humidity

• They are inactivated by UV light

• Engineering of poxvirus vectors–transgenes can be inserted into silent regions of the vaccinia genome or into nonessential genes such as the gene for thymidine kinase. In animal models, vectors with insertions into the thymidine kinase gene were 10,000 times less pathogenic.

<!––>Herpes simplex virus <!––>(HSV) vectors

• Administered in vivo.

• Little data on shedding of HSV vectors.

• Can survive on fomites (cloth or plastic) for up to 4 h and on skin for up to 2 h.

• Engineering–following infection, a viral transactivating protein induces transcription of several immediate early genes that regulate other genes required for viral replication and encapsidatation. Mutation in the immediate early genes results in a virus that is unable to replicate unless it is grown in a packaging cell line. Transgenes can be inserted into the replication deficient virus.

Non-viral vectors

• Administered in vivo.

• Transgenes are usually incorporated into a plasmid. Since plasmids do not replicate, it is unlikely that plasmid-based gene therapy vehicles would spread from person to person in the healthcare setting.

• Data from experimental studies suggest that there is no potential for replication due to recombination with any type pathogen.

What are the consequences of ignoring the key principles of gene therapy and infection control?

Patients admitted to healthcare facilities for gene therapy are intentionally exposed to engineered viral pathogens in an effort to give them an infection. Conceptually, this is the opposite of what we normally try to do in Infection Control–that is to prevent a patient from getting infected while under our care. Infection Control considerations in the gene therapy setting are mainly to prevent the cross-transmission of vector or its replication-competent reactivant to persons other than the study volunteer. Consequences of inadvertently becoming infected with a viral vector include a disease state mimicking that caused by the wild-type virus itself and the possibility of effects from transgene expression or its integration into the host genome.

Summary of current controversies.

There is currently little data on vector shedding from patients in gene therapy trials. We know that viral vectors are shed, but most clinical gene therapy trials do not report data on shedding; so the frequency and extent of shedding remain ill defined. Although published data show that shedding occurs with retroviral, adenoviral, AAV, and poxvirus, and other vectors, the amount of vector shed and its distribution in excreta or blood depend on the vector and route of administration. There is little data on the time course of shedding. Currently, the relevance and implications of shedding, which is critical to Infection Control, remain to be better elucidated.

What national and international guidelines exist related to gene therapy and infection control?

The Centers for Disease Control and Prevention (CDC), Food and Drug Administration (FDA), and National Institutes of Health (NIH) have not published guidelines for infection control in gene therapy in the clinical setting. However, FDA and NIH provide oversight for US gene therapy protocols.

• The Center for Biologics Evaluation and Research (CBER) at FDA has a primary role to ensure that manufacturers produce high-quality and safe gene therapy products and that these products are properly studied in human subjects. Researchers file an Investigational New Drug (IND) application, which must then be approved by the FDA.

• Protocols funded by the NIH are reviewed by the Recombinant DNA Advisory Committee (RAC) of the NIH Office of Biotechnology Activities (OBA). The NIH’s primary responsibility is to evaluate the quality of the science involved in human gene therapy research, and to fund laboratory and clinical research. After an initial review, the RAC can recommend a protocol undergo public review and discussion. It is strongly recommended by FDA and NIH that protocols developed by industry without federal support submit voluntarily to review by the RAC.

• At the local level, protocols must be evaluated and approved by the Institutional Review Board (IRB) and the Institutional Biosafety Committee (IBC).

What other consensus group statements exist, and what do key leaders advise?

A multidisciplinary conference sponsored by industry and the NIH was held in 1999. Representatives from basic science, industry, the FDA, the CDC, and Infection Control participated. Aspects of vector construction, safety, regulatory oversight, and infection control concerns were discussed and recommendations made. The proceedings were subsequently published. (PUBMED:11083185)

References

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