Recent technologic advances in gene transfer and cell purification have created new opportunities for therapeutic intervention in HIV-1 infection. These include the ability to purify and expand selected hematopoietic progenitors, T-cell subsets, and transduce with retroviral vectors for immunologic and hematologic reconstitution studies. Handling of HlV-1-infected cells requires the ability to purify and manipulate patient derived materials under BSL3 (biosafety level 3) conditions using good laboratory practice (GLP) guidelines and validated standard operating procedures. We have therefore developed a core BSL3 stem cell/T-cell procurement and expansion facility to function in support of clinical HIV-1 trials and basic science experiments. At present, the facility offers large (clinical) and small scale purification of CD34+ cells from all tissue sources, and hematopoietic lineage purification using immunomagnetic methods for follow-up analysis in clinical studies or for basic research projects. Other assays available in the facility include flow cytometric analysis for subset purity, colony forming assays and PCR for detection of neoR sequences. Assays for sterility, endotoxin, mycoplasma, and a PCR method for replication competent retrovirus (RCR) are available in support of gene therapy clinical studies.

The Core provides scientific advice in the following areas: i) FDA regulations governing the conduct of human gene therapy trials; ii) issues related to retroviral vectoring, including choice of envelope and producer cell line, manufacturing of viral supernatant under GMP conditions, titration assays, use of cytokine combinations, fibronectin transduction support and other ancillary products used during transduction, and iii) current guidelines and protocols available for toxicology testing in gene therapy, including assays to detect RCR and vector mediated immune responses. In support of basic gene therapy research, the Core also serves as a banking source for common retroviral packaging cell lines, reporter genes and packaging plasmids.

Fee Structure :

Recharge Process

Recharge costs will reimburse the facility for general supply and reagent costs. Recharge for procedures performed for clinical protocols such as large scale purifications or transductions will be budgeted separately. Cost of vector transduction for clinical trials will be individualized by protocol. The following are estimations of current recharge costs for commonly performed assays and procedures.

SERVICES	PRICE
Large scale (clinical) CD34 purification -Cost of Column -Technical Supplies for Separation -Technical time -Equipment	$5,825.00 $4,325.00 $600.00 $550.00 $350.00
CD 34 preparation (MACS), large preparation for cord blood and bone marrow	$1,000.00
Small scale CD34 purification (4X106) cells	$300.00
Methyl/Cellulose Colony Assays +/- G418	$110.00
Single Colony PCR	$200.00
Helper Virus Assay (PCR)	$50.00
PCR for vector seq.	$50.00
FACS Analysis(for cell subset)	$100.00
Monocyte Separation	$85.00
CD3/CD4/CD8 separation	$200.00
Granulocyte separation	$50.00
RT PCR for vector seq. in PBL	$200.00
CD3,CD4,CD8+ purification	$300.00
Purification for 2 or 3 phenotypic marker expression	$900.00
Endotoxin testing	$20.00
Mycoplasma (Gen Probe)	$20.00
Bacterial	$50.00
Fungal	$50.00
Fibronectin coated flasks (175 cm2) for retroviral transduction	$200.00

Test prices reflect cost per sample. Other services will be developed and budgeted as they are identified during the implementation of future projects. There is no charge for consultation or assistance with experimental design. Plasmids, cell lines and other gene therapy reagents will be provided at a basic cost of $35 for sample preparation and handling.