Full Text HL-94-003-B

GENE THERAPY FOR SICKLE CELL DISEASE

NIH GUIDE, Volume 22, Number 42, November 19, 1993

RFA:  HL-94-003-B

National Heart, Lung, and Blood Institute

P.T. 34

Keywords: 
  Blood Diseases 
  Gene Therapy+ 


Letter of Intent Receipt Date:  January 15, 1994
Application Receipt Date:  March 15, 1994

PURPOSE

The Division of Blood Diseases and Resources (DBDR) of the National
Heart, Lung, and Blood Institute (NHLBI), invites research grant
applications for the support of basic and applied research leading to
the development of strategies to correct and/or replace the
endogenous defective gene in sickle cell anemia.

HEALTHY PEOPLE 2000

The Public Health Service (PHS) is committed to achieving the health
promotion and disease prevention objectives of "Healthy People 2000,"
a PHS-led national activity for setting priority areas.  This Request
for Applications (RFA), Gene Therapy for Sickle Cell Disease, is
related to the priority areas of clinical prevention services,
chronic disabling conditions, and maternal and infant health.
Potential applicants may obtain a copy of "Healthy People 2000" (Full
Report:  Stock No. 017-001-00474-0) or "Healthy People 2000" (Summary
Report:  Stock No. 017-001-00473-1) through the Superintendent of
Documents, Government Printing Office, Washington, DC 20402-9325
(telephone 202-782-3238).

ELIGIBILITY REQUIREMENTS

Applications may be submitted by domestic for-profit and non-profit
organizations, public and private, such as universities, colleges,
hospitals, laboratories, units of state or local governments, and
eligible agencies of the federal government.  Awards in response to
this RFA will be made to domestic institutions only.  Applications
from minority individuals and women are encouraged.

MECHANISM OF SUPPORT

This RFA will use the National Institutes of Health (NIH) program
project grant (P01) and is a one-time solicitation.  A program
project grant accommodates the support of a research program in which
a multidisciplinary team of investigators works collaboratively in a
clearly defined area of mutual scientific interest.  In a program
project, achievement of the objectives of the research effort is
facilitated by the sharing of ideas, data, and specialized resources
such as equipment, laboratories, and clinical facilities.  An
essential requirement is the central theme toward which the total
scientific effort is directed and to which each research project
relates.  The NHLBI expects the applicant to develop the approaches
that would be used to accomplish the objectives of the proposed
research program.

The interrelated research projects included in the program should be
conducted by experienced scientists who have a variety of
disciplinary and specialty backgrounds and who are willing and able
to relate to each other so that new scientific information may be
freely exchanged and effectively utilized by others in the research
program.  The program director must be an established leader in
scientific research with demonstrated capabilities in administration.
The director is expected to demonstrate exemplary leadership by
presenting a cohesive program, focusing research efforts on the
central theme.  The quality of the written grant application and its
cohesiveness serve as an indicator of the leadership capabilities of
the director.  Meetings of participating investigators, who share and
evaluate results and new ideas, are essential to the consolidation of
the research projects into a cohesive program.  An internal advisory
committee selected from the participating investigators in the
program can be effective in assisting the program director in making
scientific and administrative decisions.  An advisory committee
composed of outside consultants can be helpful in providing
scientific and organizational advice and assisting the Principal
Investigator in maintaining and monitoring scientific progress.

The size of the program project should be carefully considered.  The
awarded program project must be composed of at least three
scientifically meritorious research projects to permit an efficacious
collaborative efforts among the participating investigators.  In
addition, no more than $1 million in direct costs in the first year
with a maximum increase of four percent in each additional year may
be requested.

An important goal of this program is to attract new and established
investigators into the field of gene therapy for sickle cell disease
by providing access to critical technologies in the form of shared
facilities and funds to pursue innovative pilot/feasibility studies.
Pilot/feasibility studies, when combined with appropriate core
support, will provide established investigators who have not
previously worked in gene therapy and new young investigators with
state-of-the-art core technologies that will enable them to be
competitive in the field.  In addition, pilot/feasibility studies
will allow investigators to pursue promising by untested
methodologies or highly novel, innovative research avenues that offer
significant results and insight.  It is anticipated that by providing
preliminary results and establishing feasibility data, these pilot
studies will lead to new R01 grant applications in the area of gene
therapy research for sickle cell disease.

Each pilot/feasibility project may request up to $60,000 in total
costs per year for a maximum of two years.  New pilot/feasibility
studies may be proposed to replace those that terminate.  If awarded,
these new pilot/feasibility studies will be subjected to the same
dollar/time limits.

Applicants, who will plan and execute their own research programs,
are requested to furnish their own estimates of the time required to
achieve the objectives of the proposed research project.  Five years
of support must be requested. At the end of the official award
period, renewal applications may be submitted for peer review and
competition for support through the regular grant program of the
NHLBI.  It is anticipated that support for the present program will
begin September 30, 1994.  Administrative adjustments in project
period/or amount of support may be required at the time of the award.
Since a variety of approaches would represent valid responses to this
announcement, it is anticipated that there will be a range of costs
among individual grants awarded.  All current policies and
requirements that govern the research grant programs of the NIH will
apply to grants awarded in connection with this RFA.

All applications submitted in response to this RFA should conform to
the policies and format described in Program Project Grant -
Preparation of the Application, NHLBI, (Revised).  A copy of this
publication may be obtained from Dr. Junius Adams at the address
listed under INQUIRIES.

FUNDS AVAILABLE

It is anticipated that for fiscal year 1994, up to two new program
project grants of up to $1,000,000 will be awarded under this
program.  It should be noted that award of grants pursuant to this
RFA is contingent upon receipt of such funds for this purpose.  It is
anticipated that the specific amount to be funded will, however,
depend on the merit and scope of the applications received and on the
availability of funds. If collaborative arrangements involve
sub-contracts with other institutions, the NHLBI Grants Operations
Branch (telephone 301-594-7436) should be consulted regarding
procedures to be followed.

RESEARCH OBJECTIVES

Sickle cell disease is a worldwide health problem and is one of the
most common inherited disorders of man.  This genetic blood disorder
is probably the best understood disease at the molecular level and
Linus Pauling coined the term "molecular disease" over forty years
ago in ascribing the abnormality to the globin portion of the
hemoglobin molecule.  Almost ten years later, the specific molecular
defect was identified as a single amino acid substitution of valine
for glutamic acid at position 6 of the beta-globin polypeptide chain.
With the advent of recombinant DNA technology, investigators were
able to further define this genetic mutation in the globin gene as a
change in the codon GAG to GTG.  The substitution of glutamic acid by
valine results in a loss of two negative charges on the surface of
the molecule making sickle hemoglobin less soluble than normal
hemoglobin upon deoxygenation.  This abnormal hemoglobin aggregates
and forms fibers within the red cells, leading to morphological
changes that subsequently affect the ability of the cells to traverse
the microvasculature, causing occlusion of these small vessels that
results in acute pain, and acute as well as chronic organ damage.  In
addition, sickle red cells are less resilient than normal cells,
leading to their early destruction and thus a chronic anemia.  This
cascade of events caused by the abnormal cell morphology affects the
structure and function of the red cells, blood flow through tissues
and organs throughout the body, and abnormal interaction of these
cells with the microvasculature.  The complex pathophysiology of this
disorder is a direct consequence of the change in morphology of red
cells containing sickle hemoglobin.  Despite the distinction of being
the first described molecular disease, there is no cure or effective
treatment currently available.

A rational therapeutic approach to this disorder would be the
replacement of the sickle beta-globin gene with a normal beta-globin
gene or to repair the sickle mutation in DNA.  Either approach would
use bone marrow stem cells manipulated in vitro to alter their
genetic makeup and subsequently reintroduce them into the patient.
Molecular studies and recent advances made at the level of the genome
that have enhanced our understanding of gene regulation and
expression, along with rapidly developing techniques of gene
transfer, have opened new avenues for the potential treatment of
genetic diseases.  The ability to insert copies of normal genes into
cells, with the production of new genes producing proteins to correct
this biochemical defect, would be a major advance in treating this
disease.  Adequate expression of the normal beta-globin gene in
patients with sickle cell anemia would be curative.

The molecular biology of globin gene expression and regulation has
supplied sufficient knowledge to make gene therapy feasible.  The
adult hemoglobin tetramer is composed of two pairs of unlike globin
chains (2 alpha and 2 beta) that are the products of distinct loci on
different chromosomes.  The alpha-globin gene cluster is located on
chromosome 16 and the beta-globin gene cluster on chromosome 11.  For
proper tetramer formation, it is essential that the alpha- and
beta-globin genes are expressed equally.  An excess or deficit of
either chain produces a thalassemia-like condition that results in
chronic anemia.  In addition to equimolar expression of alpha- and
beta-globin, control of globin gene expression is manifested in the
ontogenic changes in hemoglobin type.  Embryonic hemoglobin is the
first hemoglobin produced in the developing fetus.  It is rapidly
replaced by fetal hemoglobin (Hb F).  At birth, Hb F is already being
replaced by adult hemoglobin (Hb A), and Hb A predominates by 6
months of age.  The control of globin gene expression has been
studied in great detail.  Within and surrounding each globin gene are
groups of nucleotides that have been conserved during evolution and
are essential for proper gene expression.  Conserved DNA sequences
that are remote from the expressed genes have also been shown to play
a critical role in globin gene expression.  The most important of
these is the locus control region (LCR) that is located 3~ to the
globin genes.  The LCR is characterized by at least four Dnase
hypersensitive sites containing tissue specific consensus sequences.
In addition to the LCR, there are other consensus sequences located
throughout the globin gene clusters that are known to bind trans
acting protein factors that are critical for globin gene expression.
The best characterized erythroid-specific transcription factor,
GATA-1, is named for the tetranucleotide to which it binds.

The discovery of the LCR had great impact on the possibility of
achieving tissue specific, high level expression of globin genes
introduced ex vivo.  When "mini gene" constructs containing either
the human beta- or gamma-globin genes ligated to a portion of the LCR
were introduced into fertilized mouse ova, transgenic mice were
produced that expressed the human beta-globin gene at normal levels
only in erythroid cells and only in adults.  Similarly, the exogenous
gamma-globin gene was expressed with similar tissue specificity, but
only in the fetus.  Thus, it is possible for inserted globin genes to
be expressed normally with correct tissue and developmental
specificity independent of the site of integration of these genes.

Although normal expression of globin genes is achievable that is
independent of the site of integration, there remain two major
obstacles to being able to insert a normal globin gene in the
position of beta-S-globin gene.  (1) A mechanism is needed to
down-regulate or ablate the expression of the beta-S-globin gene.  If
the endogenous gene and the exogenous gene are co-expressed, there
would be an excess of beta-globin synthesis and an alpha-thalassemia
would ensue.  (2) If beta-globin genes are randomly inserted into the
genome, it is possible that the exogenous gene will integrate into an
unrelated gene that is critical and inactivate it.  A method of site
specific recombination has been developed in which the exogenous gene
can only insert into its endogenous counterpart.  This procedure
inactivates the endogenous gene and effectively removes any
possibility of insertional mutagenesis.  This method is currently
very inefficient and further research and development is necessary
for application to gene therapy for sickle cell disease.

Another area that needs further research is the production of a safe
and efficient vector for gene transfer.  At present, modified
retrovirus vectors appear to be the most efficacious method for
delivering gene to the target cell.  These vectors have been used
with great success in mice, but successful gene transfer and
persistent expression has been difficult to accomplish in larger
animals.  However, longer co-culture of the bone marrow with vector
producing cells has allowed the successful transfer of genes into
primate bone marrow.  Another problem has been recombination events
that return the vectors to replication competent viruses.  This
problem has been largely overcome by more refined packaging
techniques.  The use of adeno associated virus (AAV) is also of
interest.  The findings that AAV is not pathogenic to humans, has
wide host range, and integrates easily into the host genome make this
vector potentially attractive for gene therapy.

Another promising approach to the amelioration of the clinical
expression of sickle cell disease by gene therapy involves the
augmentation of fetal hemoglobin synthesis.  Since it is adult
hemoglobin that is affected in sickle cell disease, its replacement
by fetal hemoglobin would be of obvious clinical benefit.
Furthermore, fetal hemoglobin inhibits the polymerization of sickle
hemoglobin, the essence of the pathophysiology of sickle cell
disease.  It has long been known that replacement of adult hemoglobin
by fetal hemoglobin would not have any adverse effects, because
individuals with hereditary persistence of fetal hemoglobin, where
fetal hemoglobin persists into adult life, are clinically normal.
Since the discovery that the chemotherapeutic agent 5-azacytidine
could dramatically increase the levels of fetal hemoglobin in anemic
individuals, several other compounds without the undesirable side
effects of 5-azacytidine have also been shown empirically to augment
fetal hemoglobin synthesis.  These agents are not universally
applicable and they require lifelong treatment.  However, the
impressive advances in basic research discussed above on globin gene
regulation have suggested other means to achieve this goal with a
higher degree of efficiency.  The discovery of the DNA sequences and
protein factors that are responsible for the switch from fetal to
adult hemoglobin strongly suggest that this switch can be almost
completely reversed, resulting in the replacement of sickle
hemoglobin with fetal hemoglobin, which would cure the disease.

An area of research that would benefit all aspects of gene therapy
for sickle cell disease is the development of an improved animal
model of sickle cell disease.  Various strategies have been employed
to create a transgenic mouse model of sickle cell disease including
the crossing of mice carrying the human beta-S-globin gene with mice
with beta+-thalassemia, the inclusion of the human alpha-globin gene,
and the design of "super sickling" globin genes that contain the
mutations for Hb S as well as those for Hb Antilles and/or D Punjab.
Although these approaches have improved the transgenic mouse model,
further work is necessary to duplicate sickle cell disease in the
mouse.  Improved animal models would be useful for testing strategies
of homologous recombination, increasing fetal hemoglobin synthesis,
and determining the amount of synthesis from the transfected globin
gene that is necessary to ameliorate sickling.

From the preceding discussion, it is obvious that much effort is
directed toward development of strategies for gene insertion into
hematopoietic progenitor cells in human bone marrow that may be
directly applicable to the treatment of sickle cell disease.  The
genetic elements necessary for high level globin gene expression in
erythroid cells have now been identified so that tissue specific
expression at a level adequate to alter hemoglobin composition is
achievable.  Currently, the major difficulty in achieving gene
transfer is that vectors capable of transferring globin genes with
the required regulatory elements into sufficient numbers of
hemopoietic progenitor cells have yet to be developed.  However,
advances in this direction continue to occur, and the replacement of
the sickle hemoglobin gene by gene transfer continues to offer the
most promising cure for all sickle cell disease patients.  Other
modalities such as bone marrow transplantation may be applicable to a
proportion of patients with variable risks.  Long term
pharmacological manipulation of fetal hemoglobin synthesis may also
be of benefit to a large proportion of patients.  Nonetheless,
successful gene transfer would be more broadly applicable.

Sickle cell disease is an excellent candidate for gene therapy for
several reasons:  (1) the human hemoglobin molecule and the globin
gene complex that is responsible for its production represent the
best known and most studied molecular system in man; (2) the gene for
sickle hemoglobin is only active in hematopoietic tissue, and this
tissue is easily accessible as bone marrow for treatment outside of
the body; (3) the current state of technology is such that gene
therapy for sickle cell disease is attainable; (4) although the
development of gene therapy for sickle cell disease would require a
significant initial investment, it would provide a cure for this
debilitating, chronic disease that consumes a significant portion of
health care costs in the United States; (5) the development of a cure
for sickle cell disease would represent a significant improvement of
health care to an underserved minority population in this country.

The following are examples of the type of research approaches that
would be responsive to the program:

o  Improvement of the efficiency of transfection of hematopoietic
stem cells by augmenting the number of stem cells available for
transfection and increasing the efficiency of the vector.

o  Introduction of a selective advantage to stem cells that allow
successful competition with endogenous stem cell for proliferation,
self-renewal, and differentiation in vivo

o  Development of new strategies for the introduction of exogenous
genes into stem cells with the goal of obtaining therapeutically
useful levels of expression and stability of the transferred gene.

o  Development of more efficient methods for gene insertion by
homologous recombination.

o  Development of methods to silence or attenuate the expression of
the endogenous beta-S-globin gene

o  Development of improved animal models to assess the efficacy of
approaches

o  An important goal of this program is to attract new and
established investigators into the field of gene therapy for sickle
cell disease by providing access to critical technologies (in the
form of shared core facilities described above) and funds to pursue
innovative pilot/feasibility studies.  Pilot/feasibility studies will
enable established investigators who did not previously work in gene
therapy and new investigators with state-of-the-art core technologies
that will enable them to be competitive in the field.  In addition,
pilot/feasibility studies will allow investigators to pursue
promising but untested innovative research in gene therapy.

These approaches are meant to serve only as examples of the types of
research projects that would be responsive to the goals of this
solicitation.  Investigators are encouraged to develop and propose
their own innovative approaches.

SPECIAL REQUIREMENTS

Upon initiation of the program, the NHLBI will sponsor annual
meetings to encourage the exchange of information among investigators
who participate in this program.  In the preparation of the budget
for the grant application, applicants should request additional
travel funds for one meeting each year to be held in Bethesda,
Maryland.  Applicants should also include a statement in the
applications indicating their willingness to participate in such
meetings.

STUDY POPULATIONS

SPECIAL INSTRUCTIONS TO APPLICANTS REGARDING IMPLEMENTATION OF NIH
POLICIES CONCERNING INCLUSION OF WOMEN AND MINORITIES IN CLINICAL
RESEARCH STUDY POPULATIONS

NIH policy is that applicants for NIH clinical research grants and
cooperative agreements will be required to include minorities and
women in study populations so that research findings can be of
benefit to all persons at risk of the disease, disorder or condition
under study; special emphasis should be placed on the need for
inclusion of minorities and women in studies of diseases, disorders
and conditions which disproportionately affect them.  This policy is
intended to apply to males and females of all ages.  If women or
minorities are excluded or inadequately represented in clinical
research, particularly in proposed population-based studies, a clear
compelling rationale should be provided.

The composition of the proposed study population must be described in
terms of gender and racial/ethnic group.  In addition, gender and
racial/ethnic issues should be addressed in developing a research
design and sample size appropriate for the scientific objectives of
the study.  This information must be included in the form PHS 398
(rev. 9/91) in Sections 1-4 of the Research Plan AND summarized in
Section 5, Human Subjects.  Applicants are urged to assess carefully
the feasibility of including the broadest possible representation of
minority groups.  However, NIH recognizes that it may not be feasible
or appropriate in all research projects to include representation of
the full array of United States racial/ethnic minority populations
(i.e., Native Americans, Blacks, and Hispanics).  The rationale for
studies on single minority population groups should be provided.

For the purpose of this policy, clinical research includes human
biomedical and behavioral studies of etiology, epidemiology, (and
preventive strategies), diagnosis, or treatment of diseases,
disorders or conditions, including but not limited to clinical
trials.

The usual NIH policies concerning research on human subjects also
apply.  Basic research or clinical studies in which human tissues
cannot be identified or linked to individuals are excluded.  However,
every effort should be made to include human tissues from women and
racial/ethnic minorities when it is important to apply the results of
the study broadly, and this should be addressed by applicants.

For foreign awards, the policy on inclusion of women applies fully;
since the definition of minority differs in other countries, the
applicant must discuss the relevance of research involving foreign
population groups to the United States' populations, including
minorities.

If the required information is not contained within the application,
the application will be deferred until the information is provided.

Peer reviewers will address specifically whether the research plan in
the application conforms to these policies.  If the representation of
women or minorities in a study design is inadequate to answer the
scientific question(s) addressed AND the justification for the
selected study population is inadequate, it will be considered a
scientific weakness or deficiency in the study design and will be
reflected in assigning the priority score to the application.

All applications for clinical research submitted to NIH are required
to address these policies.  NIH funding components will not award
grants or cooperative agreements that do not comply with these
policies.

LETTER OF INTENT

Prospective applicants are asked by submit, by January 15, 1994, a
letter of intent that includes a descriptive title of the proposed
research, the name, address, and telephone number of the Principal
Investigator, the identities of other key personnel and participating
institutions, and the number and title of the RFA in response to
which the application may be submitted.  Such letters are requested
only for the purpose of providing an indication of the number and
scope of applications to be received; therefore their receipt is
usually not acknowledged.  A letter of intent is not binding, and it
will not enter into the review of any application subsequently
submitted, nor is it a necessary requirement for the application.

This letter of intent is to be sent to:

Acting Chief, Centers and Special Projects Review Section
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
Westwood Building, Room 553
Bethesda, MD  20892
Telephone:  (301) 594-7448
FAX:  (301) 594-7407

APPLICATION PROCEDURES

Applications are to be submitted on the research grant application
form PHS 398 (rev. 9/91).  This form is available in an applicant
institution's office of sponsored research and from the Office of
Grants Information, Division of Research Grants, National Institutes
of Health, 5333 Westbard Avenue, Room 449, Bethesda, MD 20892,
telephone (301) 435-0714.  All applications must conform to the
format and policies described in Program Project Grant - Preparation
of the Application NHLBI (revised).  In addition, ensure that the
points identified in the section on REVIEW CONSIDERATIONS are
fulfilled.

Applicants from institutions that have a General Clinical Research
Center (GCRC) funded by the NIH National Center for Research
Resources may wish to identify the GCRC as a resource for conducting
the proposed research.  If so, a letter of agreement from either the
GCRC program director or Principal Investigator could be included
with the application.

The RFA label available in the PHS 398 application kit must be
affixed to the bottom of the face page of the original copy of the
application.  Failure to use this label could result in delayed
processing of the application such that it may not reach the review
committee in time for review.  To identify the application as a
response to this RFA, check "YES" on Item 2A of page 1 of the
application and enter the title and RFA number:  GENE THERAPY FOR
SICKLE CELL DISEASE:  NHLBI RFA HL-94-003-B.

Send or deliver the completed application and three signed, exact
photocopies of it to the following, making sure that the original
application with the RFA label attached is on top:

Division of Research Grants
National Institutes of Health
Westwood Building, Room 240
Bethesda, MD  20892**

Send an additional two copies of the application to the Acting Chief,
Centers and Special Projects Review Section at the address listed
under LETTER OF INTENT.  It is important to send these two copies at
the same time as the original and three copies are sent to the
Division of Research Grants.  Otherwise the NHLBI cannot guarantee
that the application will be reviewed in competition for this RFA.

Applications must be received by March 15, 1994.  If an application
is received after that date, it will be returned to the applicant
without review.  The Division of Research Grants (DRG) will not
accept any application in response to this announcement that is
essentially the same as one currently pending initial review, unless
the applicant withdraws the pending application.  The DRG will not
accept any application that is essentially the same as one already
reviewed.  This does not preclude the submission of substantial
revisions of applications already reviewed, but such applications
must include an introduction addressing the previous critique.

REVIEW CONSIDERATIONS

Upon receipt, applications will be reviewed for completeness by the
DRG and responsiveness by the NHLBI.  Incomplete applications will be
returned to the applicant without further consideration.  If the
application is not responsive to the RFA, NHLBI staff will contact
the applicant to determine whether to return the application to the
applicant or submit it for review in competition with unsolicited
applications at the next review cycle.

Applications may be triaged by an NHLBI peer review group on the
basis of relative competitiveness.  The NIH will withdraw from
further competition those applications judged to be non-competitive
for award and notify the applicant Principal Investigator and
institutional official.  Those applications judged to be competitive
will undergo further scientific merit review.  Those applications
that are complete and responsive will be evaluated in accordance with
the criteria stated below for scientific/technical merit by an
appropriate peer review group convened by the Division of Extramural
Affairs, NHLBI.  The second level of review will be provided by the
National Heart, Lung, and Blood Advisory Council.

Review Criteria

The factors to be considered in the evaluation of scientific merit of
each application will be similar to those used in the review of
traditional research grant applications, including the novelty,
originality, and feasibility of the approach; the training,
experience and research competence of the investigators; the adequacy
of the experimental design; the suitability of the facilities; and
the appropriateness of the requested budget to the work proposed.
The major factors to be considered in the evaluation of program
project applications will include:

o  The significance of the proposed program including its potential
for successfully addressing the primary goal of gene therapy for
sickle cell disease.

o  The proposed program must adhere to the clearly defined central
theme of gene therapy for sickle cell disease to which each component
project relates and to which each investigator contributes.

o  The proposed research should represent new directions and
opportunities and demonstrate the development of significant key
alliances among investigators in the diverse areas of scientific
expertise (e.g., molecular biology, virology, cell biology, animal
models, stem cell biology, and pathology) required for further
advancement of sickle cell gene therapy research.  This RFA will also
provide the infrastructure necessary to foster such collaborative
efforts by establishing appropriate cores, such as microbiology,
vector development, cell biology, and animal model facilities.  In
addition to stimulating collaboration, these core facilities must be
designed to enhance and extend the effectiveness of the gene therapy
program by ensuring access to new biomedical research services and
specialized instrumentation, avoiding duplication of expensive effort
and increasing quality control.

o  The scientific merit of the proposed component projects, including
the originality and feasibility of the approach, the adequacy of the
experimental design, and the relevance to the central theme of the
program.

o  The quality and commitment of senior scientific leadership and
their experience and ability to successfully integrate basic,
applied, and clinical research.

o  The quality and commitment of a qualified group of project
investigators  from several scientific disciplines with the
experience, training, and abilities to successfully direct each
project and core unit.

o  The physical and intellectual resources and environment in which
the participating laboratories will operate and interact, as well as
the supportive nature and commitment of the sponsoring
institution(s).

o  Scientific merit of any proposed pilot/feasibility studies and the
quality of the internal and external review mechanisms established by
the parent institution to evaluate the scientific merit of the
initial and subsequently selected pilot/feasibility studies,
including a detailed plan for maintaining oversight and review of
on-going pilot/feasibility studies and for providing written
documentation of these actions, copies of which will be forwarded to
the NHLBI program official.

o  The proposed program must include a plan to ensure close
interaction among all participants and the communication of ideas and
results.

o  The appropriateness of the requested budget for the proposed
program.

AWARD CRITERIA

The anticipated date of award is September 30, 1994.  Funding
decisions will be made on the basis of scientific and technical merit
as determined by peer review, program needs and balance, and the
availability of funds.

INQUIRIES

Written and telephone inquiries concerning this RFA are encouraged.
The opportunity to clarify any issues or questions from potential
applicants is welcome.

Inquiries regarding programmatic issues may be directed to:

Dr. Junius G. Adams, III
Division of Blood Diseases and Resources
National Heart, Lung, and Blood Institute
Federal Building, Room 508
Bethesda, MD  20892
Telephone:  (301) 496-6931
FAX:  (301) 402-4843

For fiscal and administrative matters, contact:

Ms. Jane R. Davis
Blood Diseases and Resources Grants Management Section
National Heart, Lung, and Blood Institute
Westwood Building, Room 4A11
Bethesda, MD  20892
Telephone:  (301) 594-7436
FAX:  (301) 594-7492

AUTHORITY AND REGULATIONS

The programs of the Division of Blood Diseases and Resources, NHLBI,
are described in the Catalog of Federal Domestic Assistance number
93.839.  Awards will be made under the authority of the Public Health
Service Act, Section 301 (42 USC 241) and administered under PHS
grants policies and Federal regulations, most specifically 42 CFR
Part 52 and 45 CFR Part 74.  This program is not subject to the
intergovernmental review requirements of Executive Order 12372, or to
Health Systems Agency Review.

Bibliography

Bank A, Markowitz D, Lerner N:  Gene transfer:  A potential approach
to gene therapy for sickle cell disease. Ann NY Acad Sci 565:37-43,
1989.

Bender MA, Gelinas RE, Miller AD:  A majority of mice show long-term
expression of a human beta-globin gene after retroviral transfer into
hematopoietic stem cells. Mol Cell Biol 9:1426-1434, 1989.

Bodine DM, McDonagh KT, Brandt SI, Ney PA, Agricola B, Byrne E,
Nienhuis AW:  Development of a high-titer retrovirus producer cell
line capable of gene transfer into rhesus monkey hematopoietic stem
cells. Proc Natl Acad Sci USA 87:3738-3742, 1990.

Culliton B.:  Gene therapy begins. Science 249:1372, 1990

Dzieriak EA, Papayannopoulou T, Mulligan RC:  Lineage-specific
expression of a human beta-globin gene in a murine bone marrow
transplant recipients reconstituted with retrovirus-transduced stem
cells. Nature 33:35-41, 1988.

Karlsson S:  Treatment of genetic defects in hematopoietic cell
function by gene transfer. Blood 78:2481-2488, 1991.

Koller B, Smithies O:  Inactivation of the beta2-microglobulin locus
in mouse embryonic stem cells by homologous recombination.  Science
246:799-803, 1989.

Miller AD:  Progress toward human gene therapy. Blood 76:271-278,
1990.

Novak U, Harris EAS, Forrester W, Groudine M, Gelinas R:  High-level
beta-globin gene expression after retroviral transfer of locus
activation region-containing human beta-globin gene derivatives into
murine erythroleukemia cells. Proc Natl Acad Sci USA 87:3386-3390,
1990.

Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlaputi RS:
Insertion of DNA sequences into the human chromosomal beta-globin
locus by homologous recombination. Nature 317:230-234, 1985.

Sorrento B, Ney P, Bodine D, Nienhuis AW:  A 46 base pair enhancer
sequence within the locus activating region is required for induced
expression of the gamma-globin gene during erythroid differentiation.
Nucleic Acids Res 18:2721-2731, 1990.

Steinberg MH:  Prospects of gene therapy for hemoglobinopathies.  Am
J Med Sci 302:298-303, 1991.

.

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