This is a non-randomized clinical trial of gene transfer using a self-inactivating, insulated, lentiviral gene transfer vector to treat 23 patients with X-linked severe combined immunodeficiency (XSCID, also called SCID-X1) who are between 2 and 40 years of age; who do not have a tissue matched sibling who can donate bone marrow for a transplant; who may have failed to obtain sufficient benefit from a previous half-tissue matched bone marrow transplant; and who have clinically significant impairment of immunity. A patient s own precursor cells (also called blood stem cells) that give rise in the marrow to blood and immune cells will have been or will be collected from the patient s blood or bone marrow. A patient will not proceed to gene transfer treatment in this protocol until there are at least 3 million blood stem cells per kilogram body weight collected from the patient.

At the NIH the patient blood stem cells will be cells collected previously under NIH protocol 94-I-0073 or collected on this protocol. In most cases the harvested blood stem cells are put into frozen storage before use in this protocol. When the patient enrolled in this protocol has the required number of blood stem cells harvested, then the patient s blood stem cells will be grown in tissue culture and exposed to the lentiviral gene transfer vector containing the corrective gene. These gene corrected blood stem cells will be administered by vein to the patient. To increase engraftment of the corrected blood stem cells, patients will receive on 2 days before the gene transfer treatment a chemotherapy drug called busulfan at a total dose of 6 mg/kilogram body weight (3 mg/kilogram body weight/daily times 2 days) that is a little more than one- third the dose used in many standard bone marrow transplants. Patients will also be given another drug called palifermin that helps prevent the main side effect from the busulfan that is a type of inflammation the mouth, stomach and bowels called mucositis. After this treatment, patients will be monitored to see if the treatment is safe and whether their immune system improves. Patients will be followed at frequent intervals for the first 2 years, and less frequently thereafter so that the effectiveness in restoration of immune function and the safety of the treatment can be evaluated.

XSCID is a genetic disease caused by defects in common gamma chain, a protein found at the surface of immune cells called lymphocytes and necessary to their growth and function. XSCID patients cannot make T-lymphocytes necessary to fight infections, and their B-cells fail to make essential antibodies. Without normal T- and B-lymphocyte function patients develop fatal infections in infancy unless they are rescued by a bone marrow transplant from a healthy donor. The best type of transplant is from a tissue matched healthy brother or sister, but most XSCID patients do not have a tissue-matched sibling, and are treated with a transplant from a parent who is only half- matched by tissue typing. While a half-matched transplant from a parent can be life-saving for an infant with XSCID, a subset of patients fail to achieve sufficient long lasting restoration of immunity to prevent infections and other chronic problems.

Recent trials of gene transfer treatments using mouse retrovirus vectors for infants with XSCID have been performed and have demonstrated that this type of gene transfer can be an alternate approach for significantly restoring immunity to infants with XSCID. However, among the 18 infants with XSCID benefiting long-term from the gene transfer treatment, 5 developed T-lymphocyte leukemia and 1 died of this leukemia. Furthermore, when older children with XSCID were treated with gene transfer, the restoration of immunity was very much less than seen in the infants. These observations of gene transfer treatments using mouse retrovirus vectors to treat infants and older patients with XSCID suggests that safer and more effective vectors were needed, and that there also may be a need to give chemotherapy or other mode of conditioning to increase engraftment in the marrow of the gene corrected blood stem cells. Our data and other published studies suggest that lentivectors that are derived from the human immunodeficiency virus and have the properties of our highly modified vector called CL20-4i-EF1 - h >=c-OPT have a reduced interaction with nearby genes and therefore less of a tendency to activate genes that may lead to cancer formation. Also, this type of lentivector may work better at getting into blood stem cells.

The study purpose is to evaluate safety and effectiveness of lentiviral gene transfer treatment at restoring immune function to 23 XSCID patients who are 2 to 40 years of age, and have significant impairment of immunity. Early evidence for effectiveness will be defined by appearance and expansion in the circulation of the patient s own gene corrected T-lymphoc...

This is a Phase I/II non-randomized clinical trial of ex vivo hematopoietic stem cell (HSC) gene transfer treatment for X-linked severe combined immunodeficiency (XSCID, also known as SCID-X1) using a self-inactivating lentiviral vector incorporating additional features to improve safety and performance. The study will treat 23 patients with XSCID who are between 2 and 40 years of age and who have clinically significant impairment of immunity. Patients will receive a total busulfan dose of approximately 6 mg/kg/body weight (target busulfan Area Under Curve is 4500 min*umol/L/day) delivered as 3mg/kg body weight on day 1 and dose adjusted on day 2 (if busulfan AUC result is available) to achieve the target dose, to condition their bone marrow, and this will be followed by a single infusion of autologous transduced CD34+HSC. Patients will then be followed to evaluate engraftment, expansion, and function of gene corrected lymphocytes that arise from the transplant; to evaluate improvement in laboratory measures of immune function; to evaluate any clinical benefit that accrues from the treatment; and to evaluate the safety of this treatment. The primary endpoint of the study with respect to these outcomes will be at 2 years, though data relevant to these measures will be collected at intervals throughout the study and during the longer follow-up period of at least 15 years recommended by the FDA Guidance "Gene Therapy Clinical Trials - Observing Subjects for Delayed Adverse Events" http://www.fda.gov/downloads

• BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation / Guidances/Cellular and Gene Therapy
• ucm078719.pdf for patients participating in gene transfer clinical trials.

XSCID results from defects in the IL2RGgene encoding the common gamma chain (yc) shared by receptors for Interleukin 2 (IL-2), IL-4, IL-7, IL-9, IL-15 and IL-21. At birth XSCID patients generally lack or have a severe deficiency of T-lymphocytes and NK cells, while their B- lymphocytes are normal in number but are severely deficient in function, failing to make essential antibodies. The severe deficiency form of XSCID is fatal in infancy without intervention to restore some level of immune function. The best current therapy is a T-lymphocyte-depleted bone marrow transplant from an HLA tissue typing matched sibling, and with this type of donor it is not required to administer chemotherapy or radiation conditioning of the patient's marrow to achieve excellent engraftment and immune correction of an XSCID patient. However, the great majority of patients with XSCID lack a matched sibling donor, and in these patients the standard of care is to perform a transplant of T- lymphocyte depleted bone marrow from a parent. This type of transplant is called haploidentical because in general a parent will be only half- matched by HLA tissue typing to the affected child. Whether or not any conditioning is used, haploidentical transplant for XSCID has a significantly poorer prognosis than a matched sibling donor transplant. Following haploidentical transplant, XSCID patients are observed to achieve a wide range of partial immune reconstitution and that reconstitution can wane over time in some patients. That subset of XSCID patients who either fail to engraft, fail to achieve adequate immune reconstitution, or lose immune function over time suffer from recurrent viral, bacterial and fungal infections, problems with allo- or autoimmunity, impaired pulmonary function and/or significant growth failure.

We propose to offer gene transfer treatment to XSCID patients3 2 years of age who have clinically significant defects of immunity despite prior haploidentical hematopoietic stem cell transplant, and who lack an HLA-matched sibling donor. Our current gene transfer treatment protocol can be regarded as a salvage/rescue protocol.

Recent successful retroviral gene transfer treatment instead of bone marrow transplant (BMT) in Paris and London for 20 infants with XSCID has provided proof of principle for efficacy. However, a major safety concern is the occurrence of 5 cases of leukemia at 3-5 years after treatment triggered in part by vector insertional mutagenesis activation of LMO2 and other DNA regulatory genes by the strong enhancer present in the long-terminal repeat (LTR) of the Moloney Leukemia Virus (MLV)- based vector.

Furthermore, previous studies of gene transfer treatment of older XSCID patients with MLV- based vectors demonstrated the additional problem of failure of adequate expansion of gene corrected T- lymphocytes to the very high levels seen in infants. To reduce or eliminate this leukemia risk, and possibly enhance 13 performance sufficiently to achieve benefit in older XSCID patients, we have generated a lentivector with improved safety and performance features. We have generated a self-inactivating (SIN) lentiviral vector that is devoid of all viral transcription elements; that contains a short form of the human elongation factor 1a (EF1a) internal promoter to express a codon optimized yc cDNA; and that has flanking copies of the 400 base pair insulator fragment from the chicken HS4 (Omega)-globin locus to provide further protection from untoward effects on flanking cellular genes. Preclinical data from our own laboratories as well as from others support the hypothesis that our SIN lentiviral vector will be significantly less prone to activating cellular oncogenes in general, and LMO2 (the gene responsible for most cases of gene transfer-related leukemias) in particular. Furthermore, our vector, designated as CL20-4i-EF1a-hyc-OPT, has established activity for reconstituting yc expression and signaling in human lymphocyte cell lines and has achieved a high level of in vivo efficacy in treatment of XSCID mice and dogs. We also established a novel stable producer cell line to allow efficient and safe high titer production of clinical lentiviral vector, greatly facilitating conduct of this clinical trial.

Based on our previous experience in treating older patients with XSCID who are either partially haploidentical donor engrafted or who failed to engraft despite multiple attempts at haploidentical donor transplant, there appears to be a significant barrier to engraftment of autologous gene corrected CD34 stem cells and an associated failure of production of adequate numbers of gene corrected autologous lymphocytes. The targeted patients for this study may have some degree of lymphoid immunity either from donor lymphocytes or their own partially functional or autologous lymphocyes that may have played a role in the poor engraftment and function of their previous haploidential HSC transplant. Furthermore, some patients may also have some graft versus host disease as a result of previous HSC transplant. In addressing these barriers to engraftment in these XSCID patients, we will pre-treat with moderate dose (~6 mg/kg) busulfan to create space or niches in bone marrow for incoming autologous gene corrected HSCs.

We plan to treat up to 23 XSCID patients, where all patients will receive the identical conditioning, gene transfer treatment, and follow-up evaluation. Mobilized peripheral blood stem cells harvested by apheresis will be the first choice source of HSC for this study, but patients who for any reason cannot provide sufficient HSC by this method (e.g. poor mobilization, inefficient apheresis separation of HSC, or inadequate central access as needed for apheresis), will have HSC collected by bone marrow harvest. At the NIH, patients can use autologous CD34+ HSC collected previously under a separate currently IRB approved stem cell collection protocol (NIH protocol 94-I- 0073; H. Malech, PI) or they will have autologous CD34+ HSC collected via apheresis under this protocol. A patient enrolled in this protocol will not proceed to transduction of the autologous HSC or to busulfan conditioning (i.e. will not be treated with gene transfer corrected cells) until there are at least 3 x 106per kilogram body weight autologous CD34+HSC (from mobilized peripheral blood stem cell apheresis collection as method of choice, and/or by bone marrow harvest) available for gene transfer transduction.

Patients will undergo a pre-treatment evaluation of both laboratory and clinical measures of immune function. Autologous CD34+HSC will be transduced ex vivo with the VSV-G pseudotyped CL20-4i- EF1a-hyc-OPT lentivector. All patients will receive a single intravenous infusion of the washed transduced cells administered intravenously on protocol Day 0. on Days -3 and -2 patients will receive an infusion of busulfan ~3 mg/kilogram body weight/day (for a total dose of ~6 mg/kilogram body weight) as conditioning to enhance engraftment of gene corrected autologous CD34+HSC. On Days - 6, -5, -4 and 1, 2 and 3, patients will receive an infusion of Keratinocyte Growth Factor (palifermin) at 60 mg/kg/day. Palifermin at this dose and schedule is FDA approved to reduce or prevent mucositis following conditioning regimens, including those that use busulfan. Following the conditioning and gene transfer treatment, subjects will be supported through any period of cytopenia and monitored for safety and efficacy of the gene transfer treatment. Early evidence for efficacy will be defined by appearance and expansion in the circulation of autologous transduced T-lymphocytes with functional yc and improved laboratory measures of immune function in the interim evaluation of these parameters at 1 year after treatment. Endpoint evidence for efficacy at 2 years after treatment will include these same laboratory parameters measured at the 2 year time point plus evidence for clinical benefit. Evidence for safety will focus on the maintenance of polyclonality of vector marking, the lack of emergence of a dominant gene marked clone in any hematopoietic lineage, and no occurrence of either hematologic dysplasia or any leukemia or other cancer resulting from the gene transfer. The primary study endpoints for all laboratory and clinical measures of efficacy and safety will occur at 2 years after gene transfer treatment. However, data collection regarding efficacy will occur at frequent intervals during the 2 years leading up to the endpoint analysis, and long- term safety and efficacy evaluation will continue at intervals during the long-term follow-up recommended by FDA Guidance for gene transfer treatment studies.

• INCLUSION CRITERIA:
• A proven mutation in the common gamma chain gene as defined by direct sequencing of patient DNA
• HLA typing of the patient will have been performed before enrollment
• No available HLA matched sibling donor as determined before enrollment.
• Must be between 2 and 40 years of age and weigh greater than or equal to 10 kg
• If previously transplanted, must be greater than or equal to 18 months post haploidentical HSCT
• Expected survival of at least 120 days.
• Documented to be negative for HIV infection by genome PCR
• The patient must be judged by the primary evaluating physician to have a suitable family and social situation consistent with ability to comply with protocol procedures and the long-term follow-up requirements.
• Medical lab data (historical) of severe B cell dysfunction (low or absent IgG levels, failed immune response to vaccines); OR demonstrated requirement for intravenous gamma globulin (IVIG) (significant drop over 3 to 6 weeks between peak and trough IgG levels).
• Must be willing to have blood and tissue samples stored IN ADDITION, patients must satisfy the following Laboratory Criteria AND Clinical Criteria
• Participants of reproductive potential must agree to consistently use highly effective contraception throughout study participation and for at least 2 years post-treatment.

Acceptable forms of contraception are:

- For males: Condoms or other contraception with partner.

Laboratory Criteria: (greater than or equal to 1 must be present)

i. CD4+ lymphocytes: absolute number less than or equal to 50 percent of the lower limit of normal (LLN)

ii. CD4 plus CD45RA+ lymphocytes: absolute number less than or equal to 50 percent of the LLN OR T-cell receptor excision circles (TRECs)squared less than or equal to 5 percent of normal for age.

iii. Memory B Cells: absolute numberless than or equal to 50percent of LLN

iv. If serum IgM<normal for age

v. NK cells: absolute number less than or equal to 50 percent of LLN

vi. Lymphocyte proliferative response to each of 2 mitogens, phytohemagglutinin (PHA) and concanavalin A (ConA), is squared 25 percent with a normal control.

vii. Molecular spectratype analysis- absent or very oligoclonal (1-3 dominant peaks) in greater than or equal to 6 of the 24 V- Beta T-cell receptor families.

Clinical Criteria: (greater than or equal to 1 must be present):

i Infections (not including molluscum, warts or mucocutaneous candidiasis; see vii and viii below): greater than or equal to 3 significant new or chronic active infections during the 12 months preceding evaluation for enrollment.

Infections are defined as an objective sign of infection (fever greater than 38.3 degrees C [101 degrees F] or neutrophilia or pain/redness/swelling or radiologic/ultrasound imaging evidence or typical lesion or histology or new severe diarrhea or cough with sputum production). In addition to one or more of these signs/symptoms of possible infection, there also must be at least 1 of the following criteria as evidence of the attending physician s intent to treat a significant infection (a. and b.) or objective evidence for a specific pathogen causing the infection (c.)

-Treatment (not prophylaxis) with systemic antibacterial, antifungal or antiviral antibiotics greater than or equal to 14 days

OR

-Hospitalization of any duration for infection

OR

-Isolation of a bacteria, fungus, or virus from biopsy, skin lesion, blood, nasal washing, bronchoscopy, cerebrospinal fluid or stool likely to be an etiologic agent of infection

ii Chronic pulmonary disease as defined by:

-Bronchiectasis by x-ray computerized tomography

OR

-Pulmonary function test (PFT) evidence for restrictive or obstructive disease that is less than or equal to 60 percent of Predicted for Age

OR

-Pulse oximetry less than or equal to 94 percent in room air (if patient is too young to comply with performance of PFTs).

iii Gastrointestinal enteropathy:

-Diarrhea-watery stools greater than or equal to 3 times per day (of at least 3 months duration that is not a result of infection as defined in criterion above)

OR

-Endoscopic evidence (gross and histologic) for enteropathy (endoscopy will only be performed if medically indicated)

OR

-Other evidence of enteropathy or bacterial overgrowth syndrome: including malabsorption of fat soluble vitamin(s), abnormal D-xylose absorption, abnormal hydrogen breath test, evidence of protein losing enteropathy (for example increasingly high or frequent dosing of intravenous gamma globulin supplement required to maintain blood IgG level).

iv Poor nutrition: Requires G-tube or intravenous feeding supplement to maintain weight or nutrition.

v Auto- or allo-immunity: Examples must include objective physical findings that include, but are not limited to any one of alopecia, severe rashes, uveitis, joint pain with redness or swelling or limitation of movement that is not a result of infection, lupus-like lesions, and granulomas (Does not include auto- or allo-immune enteropathy which is criterion iii). Where possible and appropriate, diagnosis will be supported by histopathology or other diagnostic modality.

vi Failure to grow in height: less than or equal to 3 rd percentile for age

vii Skin molluscum contagiosum OR warts (this criterion is satisfied if molluscum consists of greater than or equal to 10 lesions or there are two or more lesions at each of two or more widely separated anatomic sites; or there are greater than or equal to 3 warts at different anatomic sites at the same time; or the patient has both molluscum and warts)

viii Mucocutaneous candidiasis (chronic oral thrush or candida esophagitis or candida intertriginous infection or candida nail infections; must be culture positive to satisfy this criterion)

ix Hypogammaglobulinemia: requires regular IgG supplementation

EXCLUSION CRITERIA:

• Any current or pre-existing hematologic malignancy
• Current treatment with any chemotherapeutic agent (becomes eligible if not on treatment for at least 3 months)
• Documented HIV-1 infection
• Documented active Hepatitis B infection
• Childhood malignancy (occurring before 18 years of age) in the patient or a first degree relative, or previously diagnosed known genotype of the subject conferring a predisposition to cancer (no DNA or other testing for cancer predisposition genes will be performed as part of the screen for this protocol)