# Presentation: 485749

## CTGTAC March 4, 2005, Topic II Update: Retrovirus Vector-Mediated Insertional TumorigenesisCarolyn A. Wilson, Ph.D.DCGT/OCTGT

## Update

- Review, Retroviral Insertional Mutagenesis
- Review and Brief Update: X-SCID Gene Therapy Clinical Trial in France
- FDA Responses (then and now)
- BRMAC, February, 2003
- Recommendations and Actions
- Detailed Update: X-SCID Gene Therapy Clinical Trial in France
- Today’s Presentations and Questions

## Gammaretroviral-Mediated Endogenous Gene Activation

- Gene Disruption

## Events Leading to BRMAC, 2003

- 11 children with X-linked SCID treated in France with autologous CD34+ cells modified ex vivo by retroviral vector encoding gamma-c (c) cDNA.
- 10/11, Clinical and laboratory evidence of engraftment and benefit
- 2/10 children with engraftment developed leukemia

-  Cavazzana-Calvo, M., et al. 2000. Science 288:669-72.

** **Hacein-Bey-Abina, et al. 2002. New England Journal of Medicine 346:1185-1193.

-  Hacein-Bey-Abina, S., et al. 2003. N Engl J Med 348:255-6; Science 302:415.

## LMO-2 Associated Clonal T-Cell Proliferation in Two Patients After Gene Therapy for SCID-X1Hacein-Bey-Abina, S., et al, 2003, Science 302:415

- Retroviral Vector:
- 1) Integrated into LMO-2
- 2) Resulted in Transcriptional
- Activation of LMO-2 locus

## January 13, 2003 Letter to Retroviral Vector Sponsors

| Category | Revise Informed Consent  | Monitor Clonality  | Clinical Hold |
| --- | --- | --- | --- |
| SCID, All Active Hematopoietic Stem Cells | Yes | Yes | Yes |
| Inactive HSC | Yes | Yes | Require to resume trial |
| All other retroviral vector clinical trials  | Yes | Yes | No, Recommend |

**January 13, 2003 Letter to Retroviral Vector Sponsors**

**All sponsors were asked to provide risk/benefit analysis. **
**CBER evaluated each response.**

**Notes:**
I will review the responses later in my talk

## New developments in X-SCID Gene Therapy Clinical Trial in France

- Resumed treating patients, May 2004 with following changes (1 patient treated to date):
- Maximum cell dose of 10 x 106 c(+) CD34+/kg
- Absence of family history of childhood cancer
- Absence of cytogenetic abnormality
- Presence of at least one infectious episode (i.e., pneumopathy, herpes infection, BCG infection, etc)
- >6 months of age
- P4: Relapse leukemia, died October, 2004
- P5, Status:  Complete remission, after completion of chemotherapy
- P10: presented with T cell lymphoproliferation

## FDA Response

- Three INDs placed on Clinical Hold
- X-SCID (2); ADA-SCID (1)
- Revise Informed Consent
- Notify IRB
- All sponsors that use retroviral vector:
- Informed of new events
- Notified IRBs

## Recommendations Actions

- BRMAC Meeting February, 2003

## Recommendations for X-SCID, IL-7 and JAK-3 deficiencies

**Unanimous Vote in Favor:**
- Only allow use of gene therapy in children with these conditions when there are no alternative therapies available

## Protocol Changes for X-SCID US INDs, Consistent with February 2003, BRMAC

| Malech | Weinberg  |
| --- | --- |
| Previous BMT with persistent T and B lymphocyte impairments
Compromised quality of life
Older, 2-20 years old
Case by case review by IRB, FDA | Absence of eligible HLA-matched sibling
High risk for haplo-identical transplant (e.g., infections)
Cell dose limit of 2 x 107 cells/kg total (~5 x 106 c+) |

- Shown with permission from Drs. Malech and Weinberg

**Notes:**
Malech:  Nov, 2003, Remove Hold
Weinbert, July, 2004, Remove Hold
Kohn, Dec, 2003, Remove Hold
Malech:  Nov, 2003, Remove Hold
Weinbert, July, 2004, Remove Hold

## ADA-SCID:  Spectrum of Viewpoints

- No fundamental biological difference in ADA-SCID to render outcome different than X-SCID
- Transgene (gamma-c) not having effect
- Cell dose lower in ADA-SCID
- X-SCID transgene product, gamma-c, has potential to be oncogenic via constitutive activation
- ADA-SCID transgene product simply metabolic
- “apples and oranges”, more potential for gamma-c to be “second hit”

## Recommendations for ADA-SCID – No consensus

- Remove Clinical Hold
- Use Gene Therapy only in children who fail PEG-ADA or fail BMT
- Only for those with no alternative therapy
- Children on PEG-ADA have disadvantage for GT, lose in vivo selection
- Remove PEG-ADA to provide selective advantage

## Protocol Changes for ADA-SCID US IND, Consistent with February 2003, BRMAC

| Kohn (ADA-SCID) |
| --- |
| Treat children >6 months old
Children will not receive PEG-ADA starting 2 weeks prior to treatment and following transplant (or were never receiving)
Busulfan treatment 5 days prior to transplant |

- Shown with permission from Dr. Kohn

**Notes:**
Kohn, Dec, 2003, Remove Hold
Note:  didn’t change BMT criteria – previously and still, only exclude patients with no HLA identical donor

## Recommendations:  All other Protocols Using Ex vivo Retroviral Vector Transduction of HSC

- Significant risk with any HSC therapy when  adequate numbers of transduced cells are infused.
- Important consideration in risk/benefit analysis
- Ask sponsors to analyze probability for their trial
- Risk not the same with other trials as with X-SCID
- Suitable when no alternative effective therapy; failure of standard therapy

## All other Retroviral Vectors:  Transducing HSC Consensus, Vote

- Reevaluate trials on hold
- Risk/benefit of GT vs. existing alternative therapies
- Case-by-case
- Appropriate informed consent documents
- 18 yes
- 1 no
- Encouraged FDA to request an analysis from sponsors of the probability of a similar adverse event occurring in their individual trial.

## FDA Implementation

- Evaluation of each sponsor’s responses to January, 2003 letter, all indications
- Informed consent revisions
- Threshold for clonality analysis
- Risk/benefit analysis

## Administrative and IND Status of 28 INDs

**Number of INDs**

**Administrative and IND Status of 28 INDs**
**Placed on Hold, January, 2003**

## 15 INDs Received Since January, 2003Using Retroviral Vector

**7**

**4**

**4**

## Other BRMAC Recommendations:  VECTOR SAFETY

- Advise investigators to develop vectors with improved safety.
- Important issues identified
- Suicide vectors
- Insulator elements
- Cell dose
- Number of integration sites

## FDA Actions:  VECTOR SAFETY

- National Toxicology Program, Collaborative Study: FDA, NIH, Chris Baum, David Emery
- Systematic approach to study effect of various parameters on risk of vector-mediated tumorigenesis:
- Delete U3 from LTR (SIN)
- Insert Insulator element in LTR
- Modulate MOI used for vector transduction
- Include both gammaretrovirus and lentivirus vectors
- Mouse model established by Chris Baum
- Analyze in study of sufficient size to give 90% CI in negative result

**Notes:**
We hope to come back to the committee at another time to review this proposed study in more detail in order to get your advice and feedback.  But we think it’s an important study that needs to be done to provide data to indicate whether such modifications will reduce the risk and to what extent

## Other Related FDA Action

**Workshop on Long-term Follow-up of Participants in Human Gene Transfer Research**
**June, 2004**
**Cosponsors:  FDA, ASGT, BIO, PhRMA**
**Workshop Summary, **_[www.asgt.org](http://www.asgt.org/)__ _

## Other BRMAC Recommendations

- FDA shouldn’t extrapolate an AE from one trial to others with same vector, target cell.
- FDA did not place non-SCID INDs on hold after news of third child with leukemia in French X-SCID trial
- Develop committee to determine number of integrants that would provide reduced risk
- FDA committee to develop statistical approach and risk assessment
- Carolyn Wilson and Dan Takefman, DCGT
- Steve Anderson, Associate Director for Risk Assessment, Office of Biostatistics and Epidemiology

## Computer Model Assumptions:Estimation of Number of Vector Integrations in LMO-2 ( 2 loci)

| Characteristic | Size (kb) | Probability Random Insertion | Probability Gene Biased Insertion* |
| --- | --- | --- | --- |
| Upstream LMO-2
-5-10 kb
-1-5 kb
- 1 kb | 
10.0 kb | 
6.66 x 10-7 | 
6.66 x 10-7
3.55 x 10-6
5.3 x 10-6 |
| LMO-2 Gene
0-1 kb
1-5 kb
5-10.8 kb | 
10.8 kb | 
6.66 x 10-7 | 
5.3 x 10-6
3.55 x 10-6
1.08 x 10-6 |
| Downstream LMO-2
11-16 kb
16-21 kb | 
10.0 kb | 
6.66 x 10-7 | 
1.08 x 10-6
6.66 x 10-7 |

- *Based on Wu, et al, 2003, Science 300:1749

**Notes:**
Probability of insertion into any 1kb region within each area
Gene biased insertion probabilities based on Wu et al

## Results of Simulation Modeling

| Vector Copies/Cell | Random Model | Gene Biased Model |
| --- | --- | --- |
| 1 | 20 | 50 |
| 5 | 100 | 250 |
| 10 | 200 | 500 |

**Number Vector Integrations in LMO-2 per 10****6**** cells**

**Probability that in a treatment of 10****6 ****cells, one or more cells with **
**vector integration in LMO-2 ****engrafts**

| Vector Copies/Cell | Random Model | Gene Biased Model |
| --- | --- | --- |
| 1 | 1/5000 | 1/2000 |
| 5 | 1/1000 | 1/400 |
| 10 | 1/500 | 1/200 |

**Notes:**
Assuming a rate of engraftment of 1/100,000 cells
Top table, values rounded to nearest 10
Bottom table, values hand-calculated from simulation results

## Update on P10 in X-SCID Gene Therapy Clinical Trial in France

## Update P10*

- Treated at 8 months of age
- 11 x 106 CD34+ c(+) cells/kg
- No clonal expansion seen at previous time point tested (2 months earlier)
- Approximately 33 months post-transplant, presented with T cell lymphoproliferation
- CD4 and CD8 positive
- Gamma-c positive
- Vector clonality and integration site under investigation
**No LMO-2 integrants were detected in earlier time points; No high expression of LMO-2 protein**
- Complete remission with first line treatment, steroids

*** ****Data By Permission, Jean-Hughes Trouvin, AFSSAPS, and Alain Fischer**** **

**Notes:**
Child developed rhinopharyngitis, associated with cervical lymphadenopathies; rhinopharyngitis resolved; adenopathies progressed, child developed wheezing
Blast cells in blood, nodes, bm
Enlarged mediastinum, causing respiratory distress
T cell proliferation

## Comparison of Patients with Leukemias

| Characteristic | P4* | P5* | P10# |
| --- | --- | --- | --- |
| Age at time of treatment | 1 month | 3 months | 9 months |
| Dose c+, CD34+ cells/kg | 18 x 106 | 20 x 106 | 11 x 106 |
| Time, Type T cell proliferation | 30 mos, +, 1 TCR | 34 mos
+, 3 TCR | 33 mos
TCRV5 |
| Retroviral Vector Integration | In 1st Intron, LMO-2 | In 5’ UTR, LMO-2 | 3 vector integrants
Sites TBD |

- *Hacein-Bey-Abina, S., et al, 2003, Science 302:415
-  # Data By Permission, Jean-Hughes Trouvin, Afssaps, and Alain Fischer

**Notes:**
P10:
T cells are immature cortical thymocytes, CD4 and CD8 pos
No chromos alterations
No lmo-2 expression detected
Rcr pending

## Status of Other Patients

| Patient | Age Treated (m) | Dose c+ CD34+/kg | Follow-up (yrs) | Status |
| --- | --- | --- | --- | --- |
| P1 | 11 | 3 x 106 | ~5.9 | Healthy |
| P2 | 7 | 5 x 106 | 5.8 | Healthy |
| P3 | 8 | 5 x 106 | GT failed* | BMT,healthy |
| P6 | 6 | 1 x 106 | 3.8 | Healthy |
| P7 | 11 | 4 x 106 | 3.7 | Healthy |
| P8 | 6 | 22 x 106 | 3.1 | Healthy |
| P9 | 7 | 1 x 106 | 3.1 | ↓T cell ; BMT, healthy |
| P11 | 8 | 5-7 x 106 | 1 month | Healthy |
| P12 | 15.5 yrs | 22 x 106 | NA | No T cell reconst. |

- *BCG vaccination, slenomegaly; c+ cells in spleen
- # Data By Permission, Jean-Hughes Trouvin, Afssaps, and Alain Fischer

## Regulatory Status of X-SCID Clinical Trial in France

- On clinical hold, request of investigators
- “...a risk of insertional mutagenesis is clearly identified today with the combination type of vector/nature of the transgene.  This risk warrants the necessity of reconsideration on the protocol and method used for the SCID-X1 gene transfer....the use of safer designed vectors is to be envisaged in an attempt to reducing the insertional mutagenesis risk.”
- 	-AFSSAPS

## Likelihood of Leukemia in SCID Patients After Bone Marrow Transplant?

## Summary Results from Immune Reconstitution of 132 SCID Patients

- 117 Haploidentical; 15 HLA-identical
- 102 survivors (62 on IVIG)
- 96 _>_1 year
- 68 _>_5 years
- 37 _>_10 years
- 30 deaths
- 24 from viral infections
- 2 from Candida sepsis
- 1 each, mitochondrial defect, nephrotic syndrome (subsequent to chemotherapy for mis-diagnosed malignancy); pulmonary hypertension
**No hematologic malignancies**

- Buckley, RH, 2004, Annu. Rev. Immunol. 22:625

## Role of c Transgene Product?

*AGAINST:*
- Hacein-Bey-Abina, S., et al, 2003
- No over-expression of c in leukemic cells of P4, P5
- No sequence changes in c transgene and entire retroviral vector in leukemic cells of P4, P5
- No constitutive activation of JAK-3
- No observed hematologic malignancies in other gene therapy clinical trials in X-SCID
*FOR:*
- Dave, U., et al, 2004, Science 303:333
- Identification of leukemia with insertion in both c and LMO-2

## Other Gene Therapy Clinical Trials in X-SCID

- Gaspar, et al, 2004, The Lancet 364:2181
- Weinberg (by permission), no children treated
- Malech (by permission), 2 children treated

## Unique Characteristics of French X-SCID Gene Therapy Clinical Trial

- Media Additives During Ex vivo culture and transduction:
- Fetal calf serum
- Higher concentration IL-3
- PEG-MGDF vs. TPO
- Transduction Conditions
- Shortest prestimulation period
- Use of amphotropic MLV envelope on retroviral vector pseudotype
- Longest time to follow-up (5 years)
***Significance??***

## Topic II:  Agenda and Questions

## Topic II:  Speakers

- Relevant Data from Animal Models:
- Dr. Cynthia Dunbar
- Dr. Utpal Dave
- Dr. Christopher Baum
- Update from Human Experience, ADA-SCID
- Dr. Donald Kohn

## Questions for Committee

## Question 1

- What incidence of leukemia in clinical trials using retroviral vector-mediated gene therapy for treatment of X-SCID meet the following?
-  “Human subjects are or would be exposed to an unreasonable and significant risk of illness or injury.”

## Question 2

- How to reduce the risk to subjects?
- Dose
- Vector Design

## Question 3

- Risk/benefit considerations:
- In ADA-SCID relative to X-SCID.
- In other clinical indications.

## Question 4

- Limit on vector copy number per cell for cells transduced ex vivo with lentivirus vector?