Full Text HL-93-15-H


NIH GUIDE, Volume 21, Number 37, October 16, 1992

RFA:  HL-93-15-H

P.T. 34

  Cardiovascular Diseases 
  Molecular Genetics 
  Animal Breed. & Facil., Scientific 

National Heart, Lung, and Blood Institute

Letter of Intent Receipt Date:  February 26, 1993
Application Receipt Date:  March 31, 1993


The objective of this solicitation is to encourage studies leading to
the application of gene targeting in rats, rabbits, and other species
that are more relevant to the study of cardiovascular diseases than
mice.  The studies may include the development, genetic manipulation,
and application of embryonic stem cells for gene targeting; the
application of transgenic techniques and the development of
transgenic animals; and the development and application of
retroviral, antisense, ribozyme, and other approaches for producing
animals of a desired genotype.


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), Cardiovascular Disease Genes in Animal
Models, is related to the priority area of heart disease and stroke.
Potential applicants may obtain a copy of "Healthy People 2000" (Full
Report:  Stock No. 017-001-00474-0 or Summary Report:  Stock No.
017-001-473-1) through the Superintendent of Documents, Government
Printing Office, Washington, DC 20402-9325 (telephone 202-783-3238).


Applications may be submitted by domestic and foreign for-profit and
non-profit organizations, public and private, such as universities,
colleges, hospitals, laboratories, units of State and local
governments, and eligible agencies of the Federal government.
Applications from minority individuals and women are encouraged.


This RFA solicits applications for the National Institutes of Health
(NIH) individual research grant (R01).  Responsibility for the
planning, direction, and execution of the proposed project will be
solely that of the applicant.  The total project period for
applications submitted in response to the present RFA may not exceed
five years.  This RFA is a one-time solicitation.  Future unsolicited
competing continuation applications will compete with all
investigator-initiated applications and be reviewed according to the
customary peer review procedures.

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.


Approximately $1.5 million in total costs will be provided for the
first year of support for the entire program.  It is anticipated that
up to six grants will be awarded under this program.  This level of
support is dependent on the receipt of a sufficient number of
applications of high scientific merit.  Although this program is
provided for in the financial plan of the National Heart, Lung, and
Blood Institute (NHLBI), awards pursuant to this RFA are contingent
upon the availability of funds for this purpose.  Administrative
adjustments in project period and/or amount of support may be
required at the time of the award.



Hypertension, atherosclerosis, and other cardiovascular diseases are
serious public health problems that accounted for 45 percent of all
deaths in the U.S. in 1988, and remain the major causes of mortality
in the U.S.  An estimated 61 million Americans have hypertension and
more than 500,000 deaths occur every year due to heart attacks alone.
One-third of these deaths occur in men and women under the age of 70.
Moreover, the estimated annual cost to the nation associated with
cardiovascular diseases is more than $40 billion.  Studies aimed at
understanding the molecular mechanisms involved in the etiology and
pathogenesis of hypertension, atherosclerosis, and other
cardiovascular diseases should lead to the development of new
preventive and therapeutic strategies with favorable consequences for
both the clinical and economic effects of these diseases.

Hypertension, atherosclerosis, and other cardiovascular diseases are
multifactorial in nature and generally involve complex interactions
between genetically determined homeostatic control mechanisms and
environmental factors.  Research progress is greatly hampered by the
lack of animal models that can be used to study normal and abnormal
processes involved in blood pressure regulation, lipoprotein
metabolism or cellular responses to injury.  Additionally, the best
available models of hypertension, atherosclerosis and other
cardiovascular diseases are confined to rats, rabbits and other
larger animals such as pigs.  These species permit detailed
physiological, pharmacological, and biochemical investigations, but
are limited by the current lack of available techniques to allow
precise and systematic alterations in their genetic composition and,
thus, to precisely define the contributions of specific genes and/or
their encoded proteins in cardiovascular disease processes.

Several techniques for specifically altering the genome have been
developed recently that allow single genes to be altered, ablated or
overexpressed.  These include the use of retroviral vectors to
incorporate exogenous DNA into the genome, antisense and ribozyme
approaches to induce translational arrest, the development of
transgenic animals that generally overexpress genes, and gene
targeting by homologous recombination of chromosomal DNA sequences
with newly introduced DNA.  Highly efficient techniques for random-
and targeted-deletional mutagenesis are also available.  In
conjunction with promoter- or gene- entrapment vectors, coupled with
inverse or reverse transcriptase-polymerase chain reaction, the genes
producing the mutant phenotypes can then be readily identified.  More
recently, a variety of transgenic approaches have also been developed
that allow conditional gene ablation and gene inactivation by
overexpression of dominant negative mutations.  Application of these
techniques thus provides the potential to produce animals of any
desired genotype, and have resulted in major new insights into our
understanding of development, cancer, immunology, and neurobiology.
However, almost all of these studies have been restricted to
investigations of mouse biology, since many well-characterized
genetic strains of mice are available, much is known about mouse
embryogenesis and its manipulation, and pluripotent embryonic stem
cells (ES cells) have been isolated and cultured.  The most powerful
of these approaches is "gene targeting" by homologous recombination.
This approach involves the use of standard recombinant DNA techniques
to introduce a desired mutation into a cloned DNA sequence of a
chosen locus.  The mutated sequence is then transferred into an ES
cell genome by gene targeting.  Microinjection of selected mutant ES
cells into blastocysts is then used to generate germ-line chimeras,
and finally, interbreeding of heterozygous siblings yields animals
homozygous for the desired mutation.

Thus, the technique of gene targeting by homologous recombination has
the potential to permit development of more precise animal models of
human disease, or to evaluate the contribution of candidate genes to
disease pathogenesis in existing animal models.  Additionally, this
technique provides a higher level of refinement and resolution in the
study of the phenotypic contributions of individual genes in the
whole animal.

A major technical difficulty in this approach, which largely limits
its use to the study of murine biology, is the lack of availability
of ES cells for species other than the mouse.  Additionally, less is
known about embryogenesis in rats, rabbits, and other mammals than in
mice; the larger species are more expensive, have longer gestation
periods and are more difficult to breed.  The availability of
genetically-defined inbred strains of rats and rabbits is also
limited.  Therefore, it is essential that cell lines be developed
from well characterized and genetically stable strains.  Other
potential problems, including the ability to select mutant ES cells
and the much higher frequency of nonhomologous vs. homologous
recombinant events, have largely been overcome by the development of
dominant positive and negative selection procedures.

Other techniques for gene manipulation, including the development of
transgenic animals, the direct microinjection of exogenous DNA into
tissues and zygotes, the use of retroviral vectors to incorporate
newly introduced DNA into the genome, and the reinfection of the germ
line with endogenous retroviruses, have been used with some success
in other species as well as the mouse.  However, the application of
these techniques to the study of cardiovascular diseases in species
other than the mouse is still very limited.  Each of these methods
has advantages and problems and all suffer from a major limitation -
the investigator cannot control where the foreign gene will be
integrated in the host DNA and, hence, cannot predetermine the defect
or alteration in function.  In addition, the inability to target a
gene to a specific chromosomal location may interfere with the
transplanted gene's ability to function, or may cause unwanted
inactivation or activation of endogenous genes.  Refinements of these
approaches, such as the use of recently developed vectors that allow
specific chromosomal locations to be targeted, should permit these
strategies of gene manipulation to be applied more widely to
investigate cardiovascular disorders in relevant species, and should
allow new animal models to be developed that more precisely mimic
human diseases.

To date, most transgenic models developed to gain insights into
cardiovascular biology and disease have focussed on phenotypic
changes resulting from overexpression of candidate genes.  Although
useful information can be obtained from such models, they are
complicated by the aberrant expression of genes in tissues other than
the target(s) of interest, and by potential alterations in cellular
function resulting from the marked overproduction of the protein
encoded by the transgene.  Gene ablation, on the other hand,
potentially provides unique opportunities to study phenotypic changes
resulting from the deletion of a single gene, in the absence of
confounding alterations due to extratarget tissue gene expression or
alterations in stoichiometry.  The focus of this RFA, therefore, is
on the development and application of techniques designed to produce
transgenic models with phenotypic changes due to precise, endogenous
genetic alterations.

Two separate task forces---the NHLBI Task Force on Research in
Hypertension (May 1991) and the NHLBI Task Force on Research in
Atherosclerosis (September 1991)---recognized the power of using
transgenic animals to study the consequences of a single gene at the
molecular, cellular, tissue, organ, and whole-animal levels.  Both
Task Force Reports also stated the critical need to develop and apply
transgenic and homologous recombination approaches to animal species
more ideal for studying the pathological processes of cardiovascular

Proposed Research

Gene manipulation offers new opportunities for the systematic
dissection of the action of genes in both health and disease.
Investigators can unravel complex biological phenomena by studying
selected events in whole organisms at the molecular, biochemical,
cellular, and physiological levels.  To date, the main applications
of gene manipulation and specifically gene targeting, have focused on
cancer, development, immunology, and neurological diseases.
Application of these technological advances to hypertension,
atherosclerosis, and cardiovascular research has, thus far, been
limited.  This RFA is designed to encourage the development and
application of gene manipulation technologies to studies in animals
such as rats or rabbits that are more amenable to physiological
manipulation than mice.  It is anticipated that these approaches will
further our understanding of normal and aberrant development,
structure, function, organization, regulation, and control of the
cardiovascular system as it pertains to human hypertension,
atherosclerosis, and other cardiovascular disorders.  Representative
areas of research include:

o  Development and use of techniques to introduce and express new
genetic information.

o  Development and use of approaches to abolish or modify the
expression of endogenous genes, such as homologous recombination and
other gene targeting techniques.

o  Development and application of novel approaches to alter
endogenous gene expression, such as antisense genes, ribozymes,
dominant negative mutations, or double knock-outs.

o  Identification of new genes and proteins involved in
cardiovascular function by insertional mutagenesis.

o  Utilization of genetic ablation techniques to unravel the complex
interactions of systems comprised of multiple cell types.

o  Isolation, characterization, and propagation of ES cells from
rats, rabbits, and other species.


Upon initiation of the program, the Division of Heart and Vascular
Diseases will sponsor periodic meetings to encourage exchange of
information among investigators who participate in this program, and
to stimulate collaboration.  Applicants must request additional
travel funds for a one-day meeting each year, most likely to be held
in Bethesda, MD.


Prospective applicants are asked to submit, by February 26, 1993, a
letter of intent that includes a descriptive title of the proposed
research, the name, address, and telephone number of the Principal
Investigator (PI), 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.

Although a letter of intent is not required, is not binding, and does
not enter into the review of subsequent applications, the information
that it contains is helpful in planning for the review of
applications.  It allows NHLBI staff to estimate the potential review
workload and to avoid conflict of interest in the review.

The letter of intent is to be sent to:

Dr. Charles L. Turbyfill
Review Branch
Division of Extramural Affairs
National Heart, Lung and Blood Institute
Westwood Building, Room 553A
Bethesda, MD  20892


The research grant application form PHS 398 (rev. 9/91) is to be used
in applying for these grants.  These forms are available at most
institutional business offices or may be obtained from the Office of
Grants Inquiries, Division of Research Grants, National Institutes of
Health, Westwood Building, Room 449, Bethesda, MD 20892, telephone
(301) 496-7447.

The RFA label available in the PHS 398 application form must be
affixed to the bottom of the face page 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.  In addition, to identify the application as a response
to this RFA, Check "YES",  enter the title, "Cardiovascular Disease
Genes in Animal Models", and the RFA number (HL-93-15-H) on line 2a
of the face page of the application.

Send or deliver a signed, typewritten original of the application,
including the Checklist, and three signed photocopies, in one package

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

Send two additional copies of the application to Dr. Charles
Turbyfill at the address listed under LETTER OF INTENT.  The NHLBI
cannot guarantee that the application will be reviewed in competition
for this RFA unless these two copies are sent at the same time as the
original and three copies are sent to the Division of Research

Applications must be received by March 31, 1993.  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.


Upon receipt, applications will be reviewed by NIH staff for
completeness and responsiveness.  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 NHLBI will withdraw from
further competition those applications judged to be non-competitive
for award and notify the applicant PI 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 NHLBI.  The second level of review will be
provided by the National Heart, Lung, Blood Advisory Council.

Review criteria for RFAs are generally the same as those for
unsolicited research grant applications:

o  the novelty, originality and feasibility of the approach and the
adequacy of the experimental design

o  the competence of the PI and collaborators to accomplish the
proposed research, and the commitment and time they will devote to
the project

o  the suitability of the facilities to perform the proposed
research, including laboratories, instrumentation, and data
management systems

o  the appropriateness of the requested budget and duration for the
proposed research

o  adequate plans for interaction and communication of information
and concepts among investigators involved in collaborative studies.


Although multidisciplinary approaches are encouraged, it is not the
intent of this announcement to solicit applications for large studies
that would encompass a variety of independent projects, i.e., program
projects.  This program will not support clinical trials or large
epidemiological studies.  In general, funds will not be provided for
the purchase and installation of expensive, new equipment.  Awards
under this announcement to foreign institutions will be made only for
research of very unusual merit, need and promise, and in accordance
with PHS policy governing such awards.

The anticipated date of award is December 1, 1993.


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

Direct inquiries regarding programmatic issues to:

Dr. Michael C. Lin
Hypertension and Kidney Diseases Branch
Division of Heart and Vascular Diseases
National Heart, Lung, and Blood Institute
Federal Building, Room 4C10
Bethesda, MD  20892
Telephone:  (301) 496-1857
FAX:  (301) 402-2044

Direct inquiries regarding fiscal and administrative matters to:

Ms. Jane Davis
Grants Operations Branch
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
Westwood Building, Room 4A15C
Bethesda, MD  20892
Telephone:  (301) 496-7257
FAX:  (301) 402-1200


This program is described in the Catalog of Federal Domestic
Assistance No. 93.837, Heart and Vascular Diseases.  Awards will be
made under the authorization of the Public Health Service Act, Title
IV, Part A (Public Law 78-410, as amended by Public Law 99-158, 42
USC 241 and 285) and administered under PHS grants policies and
Federal Regulations 42 CFR 52 and 45 CFR Part 74.  This program is
not subject to the intergovernmental review requirement of Executive
Order 12372 or Health Systems Agency review.  All current policies
and requirements that govern the research grant programs of the NIH
will apply to grants awarded under this RFA.


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