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Federal Register / Vol. 59, No. 127 / Tuesday, July 5, 1994 / Notices 
I diseases), the following questions 
I should be addressed: 
Appendix M-I-A-l-a. Why is the 
disease selected for treatment by means 
I of gene therapy a good candidate for 
|i such treatment? 
■i Appendix M-I-A-l-b. Describe the 
! natural history and range of expression 
I of the disease selected for treatment, 
i What objective and/or quantitative 
I measures of disease activity are 
I available? In your view, axe the usual 
effects of the disease predictable enough 
to allow for meaningful assessment of 
the results of gene therapy? 
Appendix M-l-A-l-c. Is the protocol 
designed to prevent all manifestations of 
the disease, to halt the progression of 
' the disease after symptoms have begun 
I to appear, or to reverse manifestations of 
I the disease in seriously ill victims? 
Appendix M-I-A-l-d. What 
I alternative therapies exist? In what 
j groups of patients are these therapies 
effective? What are their relative 
advantages and disadvantages as 
compared with the proposed gene 
therapy? 
I Appendix M-I-A-2. Transfer of DN A 
1 for Other Purposes. Appendix M-I-A- 
i 2-a. Into what cells will the 
recombinant DNA be transferred? 
Why is the transfer of recombinant 
DNA necessary for the proposed 
research? What questions can be 
answered by using recombinant DNA? 
Appendix M-I-A-2-b. What 
alternative methodologies exist? What 
are their relative advantages and 
disadvantages as compared to the use*of 
recombinant DNA? 
Appendix M-I-B. Research Design, 
Anticipated Risks and Benefits 
Appendix M-l-B-l. Structure and 
Characteristics of the Biological System. 
Provide a full description of the 
methods and reagents to be employed 
for gene delivery and the rationale for 
their use. The following are specific 
points to be addressed: 
Appendix M-I-B-l-a. What is the 
structure of the cloned DNA that will be 
used? 
Appendix M-I-B-l-a-(l). Describe 
the gene (genomic or cDNA), the 
bacterial plasmid or phage vector, and 
the delivery vector (if any). Provide 
complete nucleotide sequence analysis 
or a detailed restriction enzyme map of 
the total construct. 
Appendix M-I-B-l-a-(2). What 
regulatory elements does the construct 
contain (e.g., promoters, enhancers, 
polyadenylation sites, replication 
origins, etc.)? From what source are 
these elements derived? Summarize 
what is currently known about the 
regulatory character of each element. 
Appendix M-I-B-l-a-(3). Describe 
the steps used to derive the DNA 
construct. 
Appendix M-I-B-l-b. What is the 
structure of the material that will be 
administered to the patient? 
Appendix M-I-B-l-b— (1). Describe 
the preparation, structure, and 
composition of the materials that will be 
given to the patient or used to treat the 
patient’s cells: (i) If DNA, what is the 
purity (both in terms of being a single 
DNA species and in terms of other 
contaminants)? What tests have been 
used and what is the sensitivity of the 
tests? (ii) If a virus, how is it prepared 
from the DNA construct? In what cell is 
the virus grown (any special features)? 
What medium and serum are used? How 
is the virus purified? What is its 
structure and purity? What steps are 
being taken (and assays used with their 
sensitivity) to detect and eliminate any 
contaminating materials (for example, 
VL30 RNA, other nucleic acids, or 
proteins) or contaminating viruses (both 
replication-competent or replication- 
defective) or other organisms in the cells 
or serum used for preparation of the 
virus stock including any contaminants 
that may have biological effects? (iii) If 
co-cultivation is employed, what kinds 
of cells are being used for co- 
cultivation? What steps are being taken 
(and assays used with their sensitivity) 
to detect and eliminate any 
contaminating materials? Specifically, 
what tests are being conducted to assess 
the material to be returned to the patient 
for the presence of live or killed donor 
cells or other non-vector materials (for 
example, VL30 sequences) originating 
from those cells? (iv) If methods other 
than those covered by Appendices M- 
I-B-l-b-(l)-(i) through (iii) are used to 
introduce new genetic information into 
target cells, what steps are being taken 
to detect and eliminate any 
contaminating materials? What are 
possible sources of contamination? 
What is the sensitivity of tests used to 
monitor contamihation? 
Appendix M-I-B-l-b-(2). Describe 
any other material to be used in 
preparation of the material to be 
administered to the patient. For 
example, if a viral vector is proposed, 
w'hat is the nature of the helper virus or 
cell line? If carrier particles are to be 
used, what is the nature of these? 
Appendix M-I-B-2. Preclinical 
Studies, including Risk-Assessment 
Studies. Provide results that 
demonstrate the safety, efficacy, and 
feasibility of the proposed procedures 
using animal and/or cell culture model 
systems, and explain why the model(s) 
chosen is/are most appropriate. 
Appendix M-l-B-2-a. Delivery 
System. Appendix M-I-B-2-a-(l). 
What cells are the intended target cells 
of recombinant DNA? What target cells 
are to be treated ex vivo and returned 
to the patient, how will the cells be 
characterized before and after 
treatment? What is the theoretical and 
practical basis for assuming that only 
the target cells will incorporate the 
DNA? 
Appendix M-I-B-2-a-(2). Is the 
delivery system efficient? What 
percentage of the target cells contain the 
added DNA? 
Appendix M-I-B-2-a-(3). How is the 
structure of the added DNA sequences 
monitored and what is the sensitivity of 
the analysis? Is the added DNA 
extrachromosomal or integrated? Is the 
added DNA unrearranged? 
Appendix M— I— B— 2— a-(4). How many 
copies are present per cell? How stable 
is the added DNA both in terms of its 
continued presence and its structural 
stability? 
Appendix M-I-B-2-b. Gene Transfer 
and Expression. Appendix M-I-B-2-b- 
(1). What emimal and cultured cell 
models were used in laboratory studies 
to assess the in vivo and in vitro efficacy 
of the gene transfer system? In what 
ways are these models similar to and 
different from the proposed human 
treatment? 
Appendix M-I-B-2-b-(2). What is 
the minimal level of gene transfer and/ 
or expression that is estimated to be 
necessary for the gene transfer protocol 
to be successful in humans? How was 
this level determined? 
Appendix M-I-B-2-b-(3). Explain in 
detail all results from animal and 
cultured cell model experiments which 
assess the effectiveness of the delivery 
system (see Appendix M-I-B-2-a) in 
achieving the minimally required level 
of gene transfer and expression (see 
Appendix M-I-B— 2-b-(2)). 
Appendix M-I-B-2— b-(4). To what 
extent is expression only from the 
desired gene (and not from the 
surrounding DNA)? To what extent does 
the insertion modify the expression of 
other genes? 
Appendix M-I-B-2-b-(5). In what 
percentage of cells does expression from . 
the added DNA occur? Is the product 
biologically active? What percentage of 
normal activity results from the inserted 
gene? 
Appendix M-I-B-2-b-(6). Is the gene 
expressed in cells other than the target 
cells? If so, to what extent? 
Appendix M-I-B-2-C. Retrovirus 
Delivery Systems. Appendix M-I-B-2- 
c-(l). What cell types have been 
infected with the retroviral vector 
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