Translational Research - A Basic Science Perspective

1. Translational Research - A Basic Science Perspective • Translational research: translates basic science discoveries into clinical applications, and/or uses clinical observations to generate new research topics. • Focus is on the integration of activities from bench to bedside. • Translational research is increasingly a focus of funding agencies • Makes it possible to expand your funding base • Makes you eligible for new types of federal and, especially private, grants • Often involves new collaborations with clinical colleagues • Can involve prospective clinical trials, or extensive use of patient samples or information

2. Translational Projects • Identify a translational opportunity - this can be a project that involves clinical specimens • Identify a clinical colleague with whom to work • This can be on-site or off-site • On-site is better for grantsmanship purposes - easier to integrate. Needs to be a two-way street • Better if the basic research plays some role in selecting patient samples to be used - what types of clinical specimens? Are specimens analyzed in some way so as to rule them in or out of the study? • How does your research feed back into clinical practice and the understanding of disease?

3. Basic Grantsmanship • PI and a Co-PI - a joint R01 • Both individuals need to benefit and play an active role • Take IRB issues seriously - NIH spells out specific pieces of information that you must include regarding patient enrollment • Seek advice from senior colleagues • Prepare your grant sufficiently in advance so that you can show it to colleagues - no excuse for not doing this!

4. Expanding your funding base • Funding diversity when starting your lab • Get your first R01, but plan for your second • Have someone else read your grants • Joint R01 grants, with you as Co-PI. Generally, you can’t get your second R01 until you demonstrate productivity on your first. Joining with someone on an R01 is a way to get around this, to bring in an additional source of funding before you obtain your second grant. It also makes it much easier to write! • Program projects • Private awards and career development awards

5. Career Development (K) Awards • A series of grant programs, most designed to help young investigators, or mid career investigators • Many are appropriate for translational research • Specific requirements and guidelines may vary from institute to institute. This can be easily determined via web pages. • Competition for these is not as intense as it is for R01 and R21 grants • Many people do not know about these awards

6. K22 - NIH Career Transition Awards • The primary objective of the K22 program is to help the awardee develop a strong, independent research career. This will be accomplished by supporting outstanding postdoctoral scientists as they move to their first academic position as assistant professors. The award will ease the transition so the recipient can concentrate on establishing a viable research laboratory prior to applying for research grant support. The award is for two years; you apply as a postdoc. If the grant is awarded, you have 12 months to start an academic position, at which point the grant begins. So, you take this grant with you.

7. K23 - Mentored Patient-Oriented Research • The purpose of the Mentored Patient-oriented Research Career Development Award (K23) is to support the career development of investigators who have made a commitment to focus their research endeavors on patient-oriented research. This mechanism provides support for three to five years of supervised study and research for clinically trained professionals who have the potential to develop into productive, clinical investigators focusing on patient-oriented research. Clinically trained professionals or individuals with a clinical degree who are interested in further career development in biomedical research that is not patient-oriented, should apply for a Mentored Clinical Scientist Career Development (K08) Award

8. K24 - Midcareer Investigator Award in Patient-Oriented Research • The purpose of the K24 is to provide support for clinicians to allow them protected time (75%) to devote to patient-oriented research and to act as mentors for beginning clinical investigators. The target candidates are outstanding clinical scientists who are actively engaged in patient-oriented research. Candidates are generally within 15 years of their specialty training. Candidates must be able to demonstrate the need for a period of intensive research focus as a means of enhancing their clinical research careers and must be committed to mentoring the next generation of patient-oriented researchers.

9. K25 Mentored Quantitative Research • The K25 is meant to support investigators whose quantitative science and engineering research has thus far not been focused primarily on questions of health and disease. Examples of quantitative scientific and technical backgrounds considered appropriate for this award include, but are not limited to: mathematics, statistics, economics, computer science, imaging science, informatics, physics, chemistry, and engineering. The K25 Award supports the career development of such investigators who make a commitment to basic or clinical biomedicine, bioengineering, bioimaging or behavioral research. This award provides support for a period of supervised study and research for productive professionals with quantitative backgrounds. It is intended for investigators from the postdoctoral level to the level of senior faculty.

10. Specific Aims • One page maximum • Should be self explanatory - tells a story, not just bare bones specific aims • 3-5 Specific Aims • Straight forwards Aims with best prelim data go first, put risky aims at the end • Aims should talk to and build on one another

11. Specific Aims - provide enough information to make aims understandable • D1. Explore the role of the bridging sheet region in receptor binding and virus infection. Does this region represent a conserved coreceptor binding site, does it govern coreceptor binding affinity, and is it involved in triggering the fusion-inducing conformational changes? • D2. Examine the relationships between Env-coreceptor affinity and virus tropism, pathogenesis, and receptor density. We have developed technologies needed to rigorously measure Env-coreceptor binding constants, and can now study the relationship between this and viral tropism and pathogenicity using a panel of well characterized primary HIV, SIV, and SHIV Env proteins. • D3. Determine how many receptor binding events are needed to activate HIV and SIV Env trimers, and explore the consequences this has for synergistic neutralization of HIV by different classes of entry inhibitors. Our preliminary studies indicate that multiple receptor binding events are needed, which in turn led us to hypothesize and subsequently demonstrate that HIV can be synergistically inhibited by different classes of entry inhibitors.

12. Background and Significance • Three page maximum • Don’t forget Significance; I usually end this section with a specific paragraph on significance • Tell what is known, what is not known, why it is important to study what is not known, and how your Aims will address this • Refer to each Specific Aim, in bold face.

13. Background and SignificanceRefer to your Specific Aims Significance for vertical transmission. Vertical transmission of HIV results in the infection of thousands of children every year. Although vertical transmission frequently occurs during birth, a substantial proportion also occurs in utero. The presence of DC-SIGN and DC-SIGNR on specific cell types in the placenta (both maternal and fetal sides of the circulation) raises the possibility that these attachment factors could impact this process. We do not plan to directly test this possibility in vivo at the present time, concentrating instead on the potential role of DC-SIGN in sexual transmission as described above. However, our in vitro studies and detailed examination of DC-SIGN and DC-SIGNR expression may help guide future in vivo experiments or determine if they are even warranted. For example, which specific cell types in the placenta express DC-SIGN and DC-SIGNR (Specific Aim #4)? At what levels are these attachment factors expressed (Specific Aim #4); and are there differences in how viruses interact with these attachment factors (Specific Aim #2)? If there are differences, do these correlate with specific virus types (Specific Aim #2)? We feel that the studies proposed in this grant will provide important baseline information that will be needed to consider this question in the future.

14. Preliminary Results • 5-10 pages, in my opinion • A critical section - B/S should convince reviewers that you are proposing something that is worth doing, Prelim Results should convince them that you can actually do it • Refer to Spec Aims, noting how specific preliminary findings will make it possible to achieve your goals • Identify key technologies, reagents and collaborations; can list in Section C1 (reagents/assays)

15. Prelim Results - Provide a roadmapAnticipate reveiwer’s concerns • We have generated or obtained many of reagents that will be needed to accomplish the Specific Aims of this proposal. In addition, our long standing interests in HIV and SIV Env structure and function, virus entry, and the humoral response to virus infection means that we already have many of the ancillary reagents and techniques that will be required to fully understand DC-SIGN and DC-SIGNR function and their potential roles in virus transmission and infection in vitro and in vivo. Finally, the close-knit collaborations between our labs, other labs at Penn, and our interest and long track-record in establishing collaborations with scientists at other institutions means that some real synergy should be obtained in these studies. Also, please note that the five papers on DC-SIGN and DC-SIGNR we have submitted for publication in the last 6 months (listed in Section C11) are included as Appendix material and include much of our Preliminary data. The Preliminary Results contains the following sections: • C1. Cloning of DC-SIGN, establishment of cell lines, and preliminary functional studies. • C2. Generation of rabbit sera and monoclonal antibodies to DC-SIGN and DC-SIGNR. • C3. Relationship between expression levels and function. • C4. Expression of DC-SIGN in vitro, detected biochemically. • C5. Expression of DC-SIGN on PBDCs, detected by FACS. • C6. DC-SIGN structure-function studies. • C7. Binding and transmission functions are dissociable. • C8. Binding to DC-SIGN alters HIV-1 Env structure. • C9. DC SIGNR expression and function. • C10. General virological and receptor reagents and assays. • C11. DC-SIGN and DC-SIGNR related publications.

16. Prelim Results - Refer to Spec Aims We have found that there is considerable variability in how avidly Env proteins from different virus strains bind to their respective coreceptors. While we have rigorously examined binding constants for only a handful of Env proteins, it is clear that binding affinities can range from at least 4 nM to approximately 500 nM - a variance of two orders of magnitude . Do differences in how Env proteins interact with their coreceptors have any impact on sensitivity to entry inhibitors? A related question concerns mechanisms by which virus can acquire resistance to entry inhibitors - a very real concern given that entry inhibitors are now in clinical trials. Is there any cost associated with resistance to entry inhibitors, and can entry inhibitors be used in various combinations to limit virus evolution? These questions will be addressed in Specific Aim #3. In our preliminary studies, we have begun to examine how differences in Env-coreceptor interactions impact sensitivity to T20 and how viruses develop resistance to the CCR5 antagonist TAK779. We have also begun to survey primary virus strains for their sensitivity to a panel of entry inhibitors used singly and in combination. Finally, we have obtained sufficient quantities of T20 and T1249 (a more potent derivative of T20) from Trimeris, AMD3100 from Anormed, and TAK779 for the experiments described in Specific Aim #3.

17. Experimental Design • Many ways to do this; different reviewers look for different things • If you are a younger investigator, place more emphasis on how you will do things, have sections in possible outcomes and pitfalls • Don’t build a house of cards - i.e. entire project, or much of it, rests on a reagent that does not presently exist • Refer back to Prelim Results

18. Specific Aims - Refer back to Prelim Results D1a. Selection of mutations and choice of Env. Initially, we will introduce 13 single amino acid substitutions in the bridging sheet region of IIIB. As shown in Section C3, we have already gotten a good start on this project, and the specific mutations are listed in Table 1. A potential problem with our approach is that the HxB gp120 binds to CXCR4 with poor affinity . By contrast, HxB-V3-BaL gp120 binds well to CCR5. Thus, while it is relatively trivial to study the impact of these mutations on CXCR4-dependent membrane fusion activity, either in the context of cell-cell fusion or virus entry assays, it is more difficult to measure their impact on gp120-CXCR4 binding. As discussed below, we should be able to accomplish this goal. However, it may prove desirable to place some of these mutations into an X4 Env protein that binds to CXCR4 with high affinity, though we have not yet identified such a protein, at least for HIV-1. This is discussed in Section D1d.

19. Surviving and Thriving at Penn • “Hands” • Students • Technicians • Postdocs • Money • Start-up • Grants • The Science • Focus; getting papers out • Resources at Penn • Collaborations, affinity groups

20. Hands - Attracting Graduate Students • Attracting students is an important part of building a lab • Your disadvantages: you are new and relatively unknown; you may not have students in your lab yet; high faculty to student ratio • Your advantages: students frequently attracted to the labs of young faculty, who are viewed as dynamic, more hands on, more accessible, the student sees the PI more • Teaching gives you an opportunity to meet students - seminar courses are more effective than lecture courses

21. Hands - Attracting Graduate Students • Consider team-teaching a seminar course with one or more faculty members; it spreads the work around; and it also makes for a more dynamic and interesting class • Be sure to give chalk talks • When you get rotation students, work with them - go over papers with them, talk to them frequently • Get involved in recruiting; see if there are T32 grants you can join

22. Technicians • There is a great pool of technicians in the Philadelphia area - lots of excellent colleges and universities • Many students want to work for 1-3 years before going to grad school or med school, and so have a reason to do well • Get to know your personnel person; know the rules; give your technician feedback, both positive and negative • If someone is not working out, you have to cut them lose; pay attention to the probationary period

23. Diversification through collaborations • You need senior author papers - little else matters. • Joint publications are OK, and are helpful, but only if you have a sufficient number of senior author papers • Avoid publishing with your previous mentors • Too many joint publications with a well known, senior scientist, is a problem, unless if you have a sufficient number of papers that do not involve that individual. • However, an effective collaboration can greatly accelerate your research, and make it easier for you to move into new areas, giving you both scientific and funding diversity.

24. Other points in getting science done • Find an affinity group - joint lab meetings • Educate yourself about the resources available at Penn; core facilities; other faculty • You have to write quickly - don’t let things drag on, don’t wait for the perfect paper • Go to meetings - outside letters will be important for tenure; stay in touch with people