PI: Li, Chengwen Title: Enhance AAV Liver Transduction with Capsid Immune Evasion

Received: 06/25/2015 FOA: PA13-302 Council: 01/2016

Competition ID: FORMS-C FOA Title: RESEARCH PROJECT GRANT (PARENT R01)

1 R01 AI117408-01A1 Dual: DK,HL Accession Number: 3838759

IPF: 578206 Organization: UNIV OF NORTH CAROLINA CHAPEL HILL

Former Number: Department: Pediatrics

IRG/SRG: GDD AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 1:

Year 2:

Year 3:

Year 4:

Year 5:

Animals: Y 

Humans: N 

Clinical Trial: N

Current HS Code: 10 

HESC: N

New Investigator: N

Early Stage Investigator: N

Senior/Key Personnel: Organization: Role Category:

Chengwen Li University of North Carolina at Chapel 

Hill

PD/PI 

Richard Samulski University of North Carolina at Chapel 

Hill

MPI

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Expiration Date: 06/30/2016

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302 . Received Date:

2015-06-25T09:02:19.000-04:00

APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R)

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5. APPLICANT INFORMATION Organizational DUNS*: 

Legal Name*: University of North Carolina at Chapel Hill

Department: Office of Sponsored Research

Division: Research

Street1*: 104 Airport Drive, CB 1350

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City*: Chapel Hill

County: Orange

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Country*: USA: UNITED STATES

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8. TYPE OF APPLICATION* If Revision, mark appropriate box(es).

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❍ Renewal ❍ Continuation ❍ Revision ❍ D. Decrease Duration ❍ E. Other (specify) :

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9. NAME OF FEDERAL AGENCY*

National Institutes of Health

10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER

TITLE:

11. DESCRIPTIVE TITLE OF APPLICANT'S PROJECT*

Enhance AAV Liver Transduction with Capsid Immune Evasion

12. PROPOSED PROJECT

Start Date* Ending Date*

04/01/2016 03/31/2021

13. CONGRESSIONAL DISTRICTS OF APPLICANT

NC-004
Contact PD/PI: Li, Chengwen 

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302 . Received Date:

2015-06-25T09:02:19.000-04:00

SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

Prefix: First Name*: Chengwen Middle Name: Last Name*: Li Suffix: 

Position/Title: Research Assistant Professor

Organization Name*: University of North Carolina at Chapel Hill

Department: Pediatrics

Division:

Street1*: 7113 Thurston Bowles;CB:7352

Street2:

City*: Chapel Hill

County: Orange

State*: NC: North Carolina

Province:

Country*: USA: UNITED STATES

ZIP / Postal Code*: 27599-7352

Phone Number*: Fax Number: (919) 966-0907 Email*: 

15. ESTIMATED PROJECT FUNDING

a. Total Federal Funds Requested*

b. Total Non-Federal Funds* $0.00

c. Total Federal & Non-Federal Funds*

d. Estimated Program Income* $0.00

16. IS APPLICATION SUBJECT TO REVIEW BY STATE

EXECUTIVE ORDER 12372 PROCESS?*

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AVAILABLE TO THE STATE EXECUTIVE ORDER 12372

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DATE:

b. NO ● PROGRAM IS NOT COVERED BY E.O. 12372; OR

❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR

REVIEW

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● I agree*

* The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.

18. SFLLL or OTHER EXPLANATORY DOCUMENTATION File Name: 

19. AUTHORIZED REPRESENTATIVE

Prefix: Dr. First Name*: Barbara Middle Name: Last Name*: Entwisle Suffix: Ph.D.

Position/Title*: Vice Chancellor for Research

Organization Name*: University of North Carolina at Chapel Hill

Department: Office of Sponsored Research

Division: Research

Street1*: 104 Airport Drive, Suite 2200

Street2: CB 1350

City*: Chapel Hill

County: Orange

State*: NC: North Carolina

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Country*: USA: UNITED STATES

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Barbara Entwisle

Date Signed*

06/25/2015

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21. COVER LETTER ATTACHMENT File Name:CoverLetter1022072232.pdf Mime Type: application/pdf
424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Cover Page----------------------------------------------------------------------------------------- 1

 Table of Contents------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

 Project Summary/Abstract(Description)----------------------------------------------------- 6

 Project Narrative------------------------------------------------------------------------- 7

 Facilities & Other Resources-------------------------------------------------------------- 8

 Equipment--------------------------------------------------------------------------------- 10

Research & Related Senior/Key Person-------------------------------------------------------------------------- 12

PHS398 Cover Page Supplement---------------------------------------------------------------------------------- 22

PHS 398 Modular Budget---------------------------------------------------------------------------------------- 24

 Personnel Justification------------------------------------------------------------------- 30

PHS 398 Research Plan----------------------------------------------------------------------------------------- 31

 Introduction------------------------------------------------------------------------------ 32

 Specific Aims----------------------------------------------------------------------------- 33

 Research Strategy------------------------------------------------------------------------- 34

 Vertebrate Animals------------------------------------------------------------------------ 46

 Select Agent Research--------------------------------------------------------------------- 49

 Multiple PI Leadership Plan--------------------------------------------------------------- 50

 Bibliography & References Cited----------------------------------------------------------- 51

 Resource Sharing Plans-------------------------------------------------------------------- 55

 

Contact PD/PI: Li, Chengwen 

Table of Contents Page 3
	
Contact PD/PI: Li, Chengwen 

OMB Number: 4040-0010

Expiration Date: 06/30/2016

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-25T09:02:19.000-04:00

Project/Performance Site Location(s)

Project/Performance Site Primary Location ❍ I am submitting an application as an individual, and not on behalf of

a company, state, local or tribal government, academia, or other type of

organization.

Organization Name: The University of North Carolina at Chapel Hill

Duns Number:

Street1*: 104 Airport Drive, CB 1350

Street2: Suite 2200

City*: Chapel Hill

County: Orange

State*: NC: North Carolina

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*: 27599-1350

Project/Performance Site Congressional District*: NC-004

File Name Mime Type

Additional Location(s)

 Page 4 
Contact PD/PI: Li, Chengwen 

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-25T09:02:19.000-04:00

OMB Number: 4040-0001

Expiration Date: 06/30/2016

RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?* ❍ Yes ● No

1.a. If YES to Human Subjects

 Is the Project Exempt from Federal regulations? ❍ Yes ❍ No

If YES, check appropriate exemption number: 1 2 3 4 5 6

If NO, is the IRB review Pending? ❍ Yes ❍ No

IRB Approval Date:

Human Subject Assurance Number

2. Are Vertebrate Animals Used?* ● Yes ❍ No

2.a. If YES to Vertebrate Animals

Is the IACUC review Pending? ● Yes ❍ No

IACUC Approval Date:

Animal Welfare Assurance Number

3. Is proprietary/privileged information included in the application?* ❍ Yes ● No

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes ● No

4.b. If yes, please explain:

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an

environmental assessment (EA) or environmental impact statement (EIS) been performed?

❍ Yes ❍ No

4.d. If yes, please explain:

5. Is the research performance site designated, or eligible to be designated, as a historic place?* ❍ Yes ● No

5.a. If yes, please explain:

6. Does this project involve activities outside the United States or partnership with international

collaborators?*

❍ Yes ● No

6.a. If yes, identify countries:

6.b. Optional Explanation:

Filename

7. Project Summary/Abstract* Abstract1022072214.pdf Mime Type: application/pdf

8. Project Narrative* Narrative1022072216.pdf Mime Type: application/pdf

9. Bibliography & References Cited Bibliography1022072258.pdf Mime Type: application/pdf

10.Facilities & Other Resources FacilitiesAndResources1022072218.pdf Mime Type: application/pdf

11.Equipment Equipment1022072219.pdf Mime Type: application/pdf

 Page 5 
ABSTRACT 

Adeno-associated virus (AAV) vector has been successfully applied in phase I clinical trials in hemophilia B 

patients with liver targeting. However, these studies have suggested that AAV capsid specific cytotoxic T 

lymphocytes (CTL) have the potential to eliminate AAV transduced hepatocytes and result in the therapeutic 

failure. Our prior studies have demonstrated that AAV capsid antigen presentation is dose-dependent and 

requires capsid ubiquitination for proteasome mediated degradation. The contamination of empty virions in 

AAV preparation inhibits transduction from full particles of AAV vectors and potentially increases the risk of 

virus capsid antigen load. In this proposal we will investigate capsid antigen presentation from AAV empty 

virions and the effect of empty particles on antigen presentation from full virus transduction (Aim 1). To 

decrease antigen presentation on AAV transduced cells for avoiding capsid specific CTL-mediated elimination, 

it has been proposed to modify the AAV capsid surface or apply proteasome inhibitors to enhance AAV 

transduction while lowering the effective dose or to escape capsid ubiquitination. We will study the effect of 

AAV mutants and proteasome inhibitors on AAV capsid antigen presentation (Aim 2). It is well-known that the 

transduction of AAV vectors in mouse models does not always translate into the human. Finally, we will 

explore the directed evolution approach combined with a rational design strategy to isolate AAV vectors with 

human hepatocyte specific tropism and the ability to evade a capsid specific CTL response in humanized mice

(Aim 3). Elucidation of AAV empty capsid antigen presentation in vivo and the development of an AAV vector 

with enhanced human liver transduction and CTL immune-evasion will allow us to design safer and more 

effective strategies that address the current clinical complications for human liver gene therapy using AAV.

 

Contact PD/PI: Li, Chengwen 

Project Summary/Abstract Page 6
	
NARRATIVE 

Having demonstrated that AAV capsid antigen presentation is dose-dependent and requires proteasome 

mediated degradation, and modification of the AAV capsid surface induces enhanced AAV transduction while 

lowering the effective dose or decreases capsid antigen presentation, we will explore to develop AAV mutants 

with the ability to evade capsid specific CTL mediated elimination and with human hepatocyte tropism. This 

study will allow us to design safer and more effective strategies for human liver gene therapy using AAV. 

 

Contact PD/PI: Li, Chengwen 

Project Narrative Page 7
	
FACILITIES AND RESOURCES 

Chengwen Li 

Laboratory: The PI has 800 sq.ft. of lab space in the Gene Therapy Center with basic lab equipment including 

a biosafety hood, incubators, Benchtop PCR (Biorad), -80 and -20 freezers, refrigerator, and various small 

equipment for molecular biology. In addition, the lab is equipped with a Perkin Elmer spectrofluorometer, 

Sorvall benchtop centrifuge. The PI also has access to all common equipment in the Gene Therapy Center’s 

2,345 sq.ft core facility including: Perkin Elmer luminometer, Gel Documentation system, complete molecular 

biology wet lab, ultra speed centrifuges, scintillation counter, sonicator, controlled temperature rooms, dark 

room, cryostat, PCR machine, incubators, cold boxes, freezers and various small equipment for general 

molecular biology, viral tissue culture room and glassware washing facility. Also available is a P3 containment 

facility. 

Animals: A virus-free animal facility for the Center is located on the ground floor of the Thurston Bowles 

building. This room is equipped with a tissue culture hood, and animal isolator and laminar flow hood. Basic 

animal care is provided by qualified animal technicians and a certified veterinarian is on duty at all times. The 

animal imaging facility is equipped with a Xenogen IVIS Lumina and a Xenogen Kinetic for live animal imaging 

studies. 

Computers: The lab has three desktop machines equipped with molecular modeling, live animal image 

analysis, statistical and data analysis software. Access to center IBM and Macintosh computers and printers, 

laptops is also available. 

Office: Over 150 square feet of administrative office space and additional office facilities is available for 

laboratory personnel with administrative and secretarial support available for all personnel in the Gene Therapy 

Center. 

Other: Support facilities on campus include the CF Center cell culture, tissue histology and mouse model core 

facilities, Lineberger Comprehensive Cancer Center, UNC Hospitals, and the School of Public Health. 

Available within these facilities are a DNA sequencing facility, Confocal and Electron Microscopy facility, Flow 

Cytometry facility, Oligonucleotide facility as well as Phosphor Imaging, Tissue Culture, Transgenic, 

Biohazard/Virus Containment and Drug Screening facilities. Off campus facilities include: NIEHS, Duke 

University, NC State University, Research Triangle Park and Wake Forest University, all a short drive away. 

 

Contact PD/PI: Li, Chengwen 

Facilities & Other Resources Page 8
	
FACILITIES AND RESOURCES 

R. Jude Samulski 

Laboratory: Total laboratory space is approximately 2,300 square feet, equipped with chemical hoods, super 

speed centrifuges (Sorvall RC5C), cryostat, PCR machine, incubators, cold boxes, freezers and various small 

equipment for general molecular biology. Also available is a P3 containment facility, 500 square feet tissue 

culture facility with five tissue culture hoods, refrigerators, water bath, dual chamber incubators, and Centra08R table top centrifuge, and a bench with a small sink and microscope dedicated to our vector production. 

WE also have access to all common equipment in the Gene Therapy Center’s 2,345 square foot core facility, 

including: complete molecular biology wet lab, ultra speed centrifuges, scintillation counter, sonicator, 

controlled temperature rooms, dark room, viral tissue culture room and glassware washing facility. 

Clinical: N/A 

Animal: A virus-free animal facility for the Center is located on the ground floor of the Thurston Bowles 

building. This room is equipped with a tissue culture hood, and animal isolator and laminar flow hood. Basic 

animal care is provided by qualified animal technicians and a certified veterinarian is on duty at all times. 

Computer: Access to departmental IBM and Macintosh computers and printers, Ethernet link to the UNC 

Chapel Hill mainframe, which includes access to Duke University, NC State and Wake Forest library 

databases. 

Office: 170 square feet of administrative office space and additional office facilities available for laboratory 

personnel with administrative and secretarial support available for all personnel in the Gene therapy Center. 

Other: Support facilities on campus include the Lineberger Comprehensive Cancer Center, UNC Hospitals, 

and the School of Public Health. Available within these facilities are a DNA sequencing facility, Confocal and 

Electron Microscopy facility, Histocompatibility, Flow Cytometry and Clinical Immunology Laboratories, 

Oligonucleotide facility as well as Phosphor Imaging, Tissue Culture, Transgenic, Biohazard/Virus Containment 

and Drug Screening facilities. Off campus facilities include: NIEHS, Duke University, NC State University, 

Research Triangle Park and Wake Forest University, all a short drive away. 

 

Contact PD/PI: Li, Chengwen 

Facilities & Other Resources Page 9
	
MAJOR EQUIPMENT 

Chengwen Li 

High and low speed centrifuges Cryostat 

Tissue Culture Hoods Perkin Elmer Thermal Cycler (PCR) 

Air Jacketed C02 Incubators Electrophoresis equipment 

Scintillation Counter High Resolution Ion Exchange Column 

Fraction Collector Branson Sonifier II 

Spectrophotometer Ranin HPCL apparatus 

Film Processor Fotodyne Gel Camera 

Walk in Warm and Cold Rooms 

 

Contact PD/PI: Li, Chengwen 

Equipment Page 10
	
MAJOR EQUIPMENT

R. Jude Samulski 

High and low speed centrifuges Cryostat 

Tissue Culture Hoods Perkin Elmer Thermal Cycler (PCR) 

Air Jacketed C02 Incubators Electrophoresis equipment 

Scintillation Counter High Resolution Ion Exchange Column 

Fraction Collector Branson Sonifier II 

Spectrophotometer Ranin HPCL apparatus 

Film Processor Fotodyne Gel Camera 

Walk in Warm and Cold Rooms New Brunswick Sci. C25 Incubator Shaker 

Wave insect cell bioreactor 

 

Contact PD/PI: Li, Chengwen 

Equipment Page 11
	
Contact PD/PI: Li, Chengwen 

OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302 . Received Date:

2015-06-25T09:02:19.000-04:00

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Project Director/Principal Investigator

Prefix:  First Name*: Chengwen Middle Name  Last Name*: Li Suffix: 

Position/Title*: Research Assistant Professor

Organization Name*: University of North Carolina at Chapel Hill

Department: Pediatrics

Division:

Street1*: 7113 Thurston Bowles;CB:7352

Street2:

City*: Chapel Hill

County: Orange

State*: NC: North Carolina

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*: 27599-7352

Phone Number*:  Fax Number: E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  MD/PhD Degree Year:  1995

File Name Mime Type

Attach Biographical Sketch*: Li_Biosketch1022072247.pdf application/pdf

Attach Current & Pending Support:

 Page 12 
Contact PD/PI: Li, Chengwen 

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302 . Received Date:

2015-06-25T09:02:19.000-04:00

PROFILE - Senior/Key Person

Prefix:  First Name*: Richard Middle Name J Last Name*: Samulski Suffix: 

Position/Title*: Dir, Gene Therapy Center, Prof

Organization Name*: University of North Carolina at Chapel Hill

Department: Pharmacology

Division:

Street1*: 7119 Thurston-Bowles;CB:7352

Street2:

City*: Chapel Hill

County: Orange

State*: NC: North Carolina

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*: 27599-7352

Phone Number*:  Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  PhD Degree Year:  1982

File Name Mime Type

Attach Biographical Sketch*: Samulski_Biosketch1022072231.pdf application/pdf

Attach Current & Pending Support:

 Page 13 
 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: LI, CHENGWEN

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: RESEARCH ASSISTANT PROFESSOR 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

Tongji Medical University, Wuhan, Hubei, China BS 07/87 Medicine

Peking Union Medical College (PUMC), China MD/PhD 07/95 Immunotherapy

Tokai University School of Medicine, Japan Postdoctoral 01/2000 Immunology, Cell 

Therapy

Gene Therapy Center, UNC at Chapel Hill, NC USA Postdoctoral 05/04 Gene Therapy, 

Immunology

A. Personal Statement 

My lab’s research has focused on the study of the immune response to adeno-associated virus (AAV) vector in 

vitro and in animal models. The ultimate goal is to develop effective strategies to evade immune response 

mediated elimination of AAV transduced target cells and escape neutralizing antibody activity. During past 

years, we have demonstrated that AAV capsids elicit a CTL response via both the classical pathway and 

cross-presentation of capsid antigens after AAV administration; capsid antigen cross-presentation is 

dependent on proteasome mediated degradation of AAV capsid and requires AAV virion escape from the 

endosome. In addition, AAV capsid antigen presentation is dose-dependent and more effective at the earlier 

period after AAV transduction. In summary, our track record, technical expertise, resources are well-suited to 

successfully achieve the goals outlined in the current proposal. We anticipate that progress made over the next 

few years will lay the groundwork to design safer and more effective AAV vectors for clinical gene therapy 

studies. 

1. Li C, Hirsch M, Asokan A, Zeithaml B, Ma H, Kafri T, Samulski RJ. Adeno-associated virus type 2 (AAV2) 

capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 

capsid in vivo. J Virol. 2007 Jul;81(14):7540-7. Epub 2007 May 2. 

2. Li C, Hirsch M, DiPrimio N, Asokan A, Goudy K, Tisch R, Samulski RJ. Cytotoxic-T-lymphocyte-mediated 

elimination of target cells transduced with engineered AAV type 2 vector in vivo. J Virol. 2009 

Jul;83(13):6817-24. 

3. Li C, He Y, Nicolson S, Hirsch M, Weinberg MS, Zhang P, Kafri T, Samulski RJ. Adeno-associated virus 

capsid antigen presentation is dependent on endosomal escape. J Clin Invest. 2013 Mar 1;123(3):1390-

401

4. He Y, Weinberg MS, Hirsch M, Johnson MC, Tisch R, Samulski RJ, Li C. Adeno-associated virus capsid 

antigen presentation is dependent on endosomal escape. Hum Gene Ther. 2013 May;24(5):545-53.

B. Position and Honors 

Positions and Employment 

2004-2010 Research Associate, The University of North Carolina at Chapel Hill, NC, 

Contact PD/PI: Li, Chengwen 

Biosketches Page 14 
2010-present Research Assistant Professor, The University of North Carolina at Chapel Hill, NC, Department 

of Pediatrics 

 

Professional Societies 

1997-2002 Member of the American Society of Hematology 

2009-present Member of American Society of Gene and Cell Therapy 

C. Contribution to Science 

1. AAV Neutralizing antibody: Gene therapy using adeno-associated virus (AAV) as a delivery vehicle has 

been successful in phase I clinical trials in patients with blood diseases and blindness. A major restriction for 

systemic AAV vector application is the high prevalence of AAV neutralizing antibodies (NAbs) in the human 

population. Over 90% of the population is naturally infected by AAV and about half still have neutralizing 

antibodies. We have recently performed a kinetics study analyzing NAb prevalence in 62 children with 

hemophilia A, and demonstrated that i) if a low titer of NAbs exists, an alternative serotype or modified capsid 

should be considered and ii) the earlier in life that vector can be administrated, the better the chance of 

avoiding the likelihood of primary wt AAV infection and natural NAb levels we observed that increases with 

age. Additionally, our studies have demonstrated that A20 antibody (which only recognizes intact AAV2 virion) 

cannot block AAV2.5 (AAV2 capsid with 5 amino acids substitution from AAV1 capsid) transduction, and no 

obvious relationship is observed between muscle transduction from mutants and inserted amino acid 

properties, or NAb titer and cross-reactivity. This result suggests that the enhanced muscle transduction and 

immune profile change from mutants with 265 insertions are related to structural changes of the mutant virion. 

a. Li C, Narkbunnam N, Samulski RJ, Asokan A, Hu G, Jacobson LJ, Manco-Johnson MJ, Monahan PE; 

Joint Outcome Study Investigators. Neutralizing antibodies against adeno-associated virus examined 

prospectively in pediatric patients with hemophilia. Gene Ther. 2012 Mar;19(3):288-94

b. Bowles DE, McPhee SW, Li C, Gray SJ, Samulski JJ, Camp AS, Li J, Wang B, Monahan PE, 

Rabinowitz JE, Grieger JC, Govindasamy L, Agbandje-McKenna M, Xiao X, Samulski RJ. Phase 1 

gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector. Mol Ther. 

2012 Feb;20(2):443-55. 

c. Li C, Diprimio N, Bowles DE, Hirsch ML, Monahan PE, Asokan A, Rabinowitz J, Agbandje-McKenna M, 

Samulski RJ. Single amino acid modification of adeno-associated virus capsid changes transduction 

and humoral immune profiles. J Virol. 2012 Aug;86(15):7752-9. 

2. Immune response to transgene: The immune response has been implicated as a critical factor in 

determining the success of gene therapy in clinical trials. Generally, the CD8+ CTL immune response is crucial 

for the specific eradication of cells displaying foreign peptides (i.e., virus- or bacteria-infected cells). Epitopes 

recognized by CTLs likely arise from primary ORFs, particularly via the degradation of newly synthesized 

proteins. However, recent studies indicate that CTL epitopes can also be encoded by non-primary ORF 

sequences and other nontraditional sources in tumor- and virus-infected cells. We have demonstrated the 

previously uncharacterized finding that a therapeutic cassette currently being used for human investigation 

displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T 

lymphocyte (CTL) response. Such unforeseen epitope generation warrants careful analysis of transgene 

sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical 

gene therapy protocols.

a. Li C, Goudy K, Hirsch M, Asokan A, Fan Y, Alexander J, Sun J, Monahan P, Seiber D, Sidney J, Sette 

A, Tisch R, Frelinger J, Samulski RJ. Cellular immune response to cryptic epitopes during therapeutic 

gene transfer. Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10770-4.

b. Mendell JR, Campbell K, Rodino-Klapac L, Sahenk Z, Shilling C, Lewis S, Bowles D, Gray S, Li C, 

Galloway G, Malik V, Coley B, Clark KR, Li J, Xiao X, Samulski J, McPhee SW, Samulski RJ, Walker 

CM. Dystrophin immunity in Duchenne's muscular dystrophy. N Engl J Med. 2010 Oct 7;363(15):1429-

37.

3. Optimization of transgene cassette: To develop AAV liver specific transgene expression, we have found 

that a small regulatory element from AAV2 integration site of the human chromosome 19 has enhancer 

 

Contact PD/PI: Li, Chengwen 

Biosketches Page 15
	
function only in liver but not in muscle. Many diseases result from accumulation of misfolded protein in the 

cytoplasm; addition of wild type gene product by gene delivery leads to improvement but may not completely 

correct the phenotype. Using alpha-1 antitrypsin (AAT) deficiency with the piZZ mutant phenotype as a model 

system, we demonstrated that a single AAV vector is able to deliver two gene products to both silence the piZZ 

transcript and restore circulating wild-type AAT expression from a resistant codon-optimized AAT (AAT-opt) 

transgene cassette. These results implicate that similar gene-therapy strategies could be considered for any 

diseases caused by accumulation of misfolded proteins. 

a. Li C, Hirsch M, Carter P, Asokan A, Zhou X, Wu Z, Samulski RJ. A small regulatory element from 

chromosome 19 enhances liver-specific gene expression. Gene Ther. 2009 Jan;16(1):43-51. 

b. Li C, Xiao P, Gray SJ, Weinberg MS, Samulski RJ. Combination therapy utilizing shRNA knockdown 

and an optimized resistant transgene for rescue of diseases caused by misfolded proteins. Proc Natl 

Acad Sci U S A. 2011 Aug 23;108(34):14258-63. 

c. Monahan PE, Sun J, Gui T, Hu G, Hannah WB, Wichlan DG, Wu Z, Grieger JC, Li C, Suwanmanee T, 

Stafford DW, Booth CJ, Samulski JJ, Kafri T, McPhee SW, Samulski RJ. Employing a gain-of-function 

factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: 

preclinical evaluation supporting an ongoing adeno-associated virus clinical trial. Hum Gene Ther. 2015 

Feb;26(2):69-81.

Complete List of Published Work in MyBibliography: 

 

http://www.ncbi.nlm.nih.gov/sites/myncbi/chengwen.li.1/bibliography/47955510/public/?sort=date&direction=as

cending 

D. Research Support 

Ongoing Research Support 

R01DK084033-03 (Li/Samulski MPI) 04/01/12-03/31/17 

NIH/NIDDK 

AAV Gene Therapy for AAT Deficiency 

The proposed studies seek to develop novel AAV vectors with improved liver-specific targeting and explore the 

ability of these vectors to escape neutralizing antibody in an AAT deficiency mouse model (PiZZ mice). 

P01 HL112761 (Samulski, PI) 2/08/2013 – 1/31/2018 

NIH NHLBI

Neutralizing antibody and AAV FIX gene therapy 

Role: Co-PI Animal Models Core 

The goal of this program is overcome the major barrier to AAV gene therapy that exists in up to 50% of the 

population who have circulating AAV neutralizing antibodies (NAb). 

 

Contact PD/PI: Li, Chengwen 

Biosketches Page 16
	
 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: SAMULSKI, RICHARD JUDE

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Director, Gene Therapy Center Professor

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

Clemson University - Clemson, SC BS 05/76 Microbiology

University of Florida – Gainesville, FL PhD 05/82 Molecular Biology

SUNY - Stony Brook, NK Postdoctoral 05/84 Microbiology

Princeton University - Princeton, NJ Postdoctoral 05/86 Molecular Biology

A. Personal Statement 

I have the expertise, leadership, and motivation necessary to successfully carry out the proposed research 

project. I have a broad background in gene therapy related to adeno-associated virus (AAV) vector, with 

specific expertise in optimization of AAV vector for clinical applications. AAV is the only known human DNA 

virus that is non-pathogenic, has broad tropism, and is able to establish both latent and lytic life cycle. Since 

our contribution of the original cloning of an infectious AAV genome, my lab has focused over 30 years on 

development of this unique virus as a gene delivery system. These efforts have led to the first long term gene 

delivery in muscle, brain and other target tissue. Development of novel AAV variants as alternative vectors and 

improvements in AAV production have allowed the lab to support the first clinical trial for gene delivery to brain 

and develop the first chimeric AAV for gene delivery for muscular dystrophy. One of my current goals is to 

continue to derive delivery systems for safe and efficient use in human gene therapy with the ultimate goal of 

facilitating the progression and translation of gene therapy research from the laboratory bench into Phase I 

clinical trials for the treatment of human disease. Recent clinical trials in patients with hemophilia B have 

demonstrated that an AAV capsid -specific CTL response eliminates AAV transduced hepatocytes resulting in 

therapeutic failure. To better understand this “rate-limiting” step, under NIH-funded grant, we observed and 

published that capsid antigen presentation in AAV transduced cells is dependent on proteasome mediated 

AAV capsid degradation as well as determined that capsid antigen presentation is dose-dependent. These 

observations laid the groundwork for our proposed research by development and testing of novel AAV vectors 

with CTL evasion potential. We have established working protocols, assays and animal models to facilitate our 

hypothesis of “evolving AAV capsid to avoid immune response in humanized liver model”.

1. Li C, Hirsch M, DiPrimio N, Asokan A, Goudy K, Tisch R, Samulski RJ. Cytotoxic-T-lymphocyte-mediated 

elimination of target cells transduced with engineered AAV type 2 vector in vivo. J Virol. 2009 

Jul;83(13):6817-24. 

2. Li C, He Y, Nicolson S, Hirsch M, Weinberg MS, Zhang P, Kafri T, Samulski RJ. Adeno-associated virus 

capsid antigen presentation is dependent on endosomal escape. J Clin Invest. 2013 Mar 1;123(3):1390-

401

3. He Y, Weinberg MS, Hirsch M, Johnson MC, Tisch R, Samulski RJ, Li C. Adeno-associated virus capsid 

antigen presentation is dependent on endosomal escape. Hum Gene Ther. 2013 May;24(5):545-53.

Contact PD/PI: Li, Chengwen 

Biosketches Page 17 
4. Shen S, Horowitz ED, Troupes AN, Brown SM, Pulicherla N, Samulski RJ, Agbandje-McKenna M, Asokan 

A. Engraftment of a galactose receptor footprint onto adeno-associated viral capsids improves transduction 

efficiency. J Biol Chem. 2013 Oct 4;288(40):28814-23

B. Position and Honors 

Positions and Employment 

1986-1992 Assistant Professor, Department of Biological Sciences, University of Pittsburgh, 

Pittsburgh PA 

1992-1993 Associate Professor, Department of Biological Sciences, University of Pittsburgh, Pittsburgh 

PA

1993-1999 Associate Professor, Department of Pharmacology, University of North Carolina, Chapel Hill 

1994-1997 Member, Recombinant DNA Advisory Committee, NIH 

1993-present Director, Gene Therapy Center, University of North Carolina, Chapel Hill, NC 

1999-present Professor, Department of Pharmacology, University of North Carolina, Chapel Hill, NC 

2003–present Founder, Acting Chief Scientific Officer, Asklepios BioPharmaceuticals, Inc., Chapel Hill, NC

 2009-2010 Vice President, American Society of Gene & Cell Therapy (ASGCT). 

2010-2011 President-Elect, American Society of Gene & Cell Therapy (ASGCT). 

 2011-2012 President, American Society of Gene & Cell Therapy (ASGCT). 

Ad hoc reviewer for National Science Foundation grant applications 

Ad hoc reviewer for March of Dimes grant applications 

Member, International Advisory Committee for the Xth International Congress of Virology 

Honors and Awards 

1990 Outstanding Young Men of America Award 

1991 University of Pittsburgh, President's Distinguished Research Award 

2008 ASGT Outstanding Achievement Award 

C. Contribution to Science 

1. AAV biology: AAV transduction involves many steps including AAV binding to the receptors or co-receptors 

on the cell surface, endocytosis, escape from the endosomes, trafficking to the perinuclear space, nucleus 

entrance, uncoating and transgene expression. A strong focus of my lab is basic understanding of AAV biology 

which has led to the first discovery of an AAV receptor (heparan sulfate) and co-recptor (αvβ5), mechanism of 

viral trafficking and genome persistence. As pioneers, we have identified several primary receptors and coreceptors as well as elucidated the mechanism of AAV trafficking intracellularly, and maintain a robust effort 

studying AAV biology with both natural and chimeric AAV capsids. 

a. Summerford C, Samulski RJ. Membrane-associated heparan sulfate proteoglycan is a receptor for 

adeno-associated virus type 2 virions. J Virol. 1998 Feb;72(2):1438-45. 

b. Summerford C, Bartlett JS, Samulski RJ. AlphaVbeta5 integrin: a co-receptor for adeno-associated 

virus type 2 infection. Nat Med. 1999 Jan;5(1):78-82.

c. Xiao PJ, Samulski RJ. Cytoplasmic trafficking, endosomal escape, and perinuclear accumulation of 

adeno-associated virus type 2 particles are facilitated by microtubule network. J Virol. 2012 

Oct;86(19):10462-73. 

d. Nicolson SC, Samulski RJ. Recombinant adeno-associated virus utilizes host cell nuclear import 

machinery to enter the nucleus. J Virol. 2014 Apr;88(8):4132-44. 

2. AAV capsid engineering: Reengineering of adeno-associated virus (AAV) isolates may yield variants with 

improved properties for clinical applications. We have performed the first clinical trial in patients with Duchenne 

muscular dystrophy using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) 

derived from a rational design strategy. The novel chimeric vector has the improved muscle transduction 

capacity of AAV1 with reduced antigenic cross-reactivity against both parental serotypes, while keeping the 

AAV2 receptor binding. Also, we observed that reengineering AAV receptors change AAV tropism and 

enhance transduction. Using an AAV evolution approach, AAV mutants can be isolated with specific tissue 

tropism and the ability to evade neutralizing antibodies. This is a major focus of the lab. 

 

Contact PD/PI: Li, Chengwen 

Biosketches Page 18
	
 

a. Li W, Asokan A, Wu Z, Van Dyke T, DiPrimio N, Johnson JS, Govindaswamy L, Agbandje-McKenna M, 

Leichtle S, Redmond DE Jr, McCown TJ, Petermann KB, Sharpless NE, Samulski RJ. Engineering and 

selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles. Mol 

Ther. 2008 Jul;16(7):1252-60.

b. Asokan A, Conway JC, Phillips JL, Li C, Hegge J, Sinnott R, Yadav S, DiPrimio N, Nam HJ, AgbandjeMcKenna M, McPhee S, Wolff J, Samulski RJ. Reengineering a receptor footprint of adeno-associated 

virus enables selective and systemic gene transfer to muscle. Nat Biotechnol. 2010 Jan;28(1):79-82. 

c. Bowles DE, McPhee SW, Li C, Gray SJ, Samulski JJ, Camp AS, Li J, Wang B, Monahan PE, 

Rabinowitz JE, Grieger JC, Govindasamy L, Agbandje-McKenna M, Xiao X, Samulski RJ. Phase 1 

gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector. Mol Ther. 

2012 Feb;20(2):443-55. 

d. Shen S, Horowitz ED, Troupes AN, Brown SM, Pulicherla N, Samulski RJ, Agbandje-McKenna M, 

Asokan A. Engraftment of a galactose receptor footprint onto adeno-associated viral capsids improves 

transduction efficiency. J Biol Chem. 2013 Oct 4;288(40):28814-23. 

3. AAV Neutralizing antibody: While AAV gene therapy continues to yield clinical results supportive of the 

hope for eventual treatment of many diseases, the presence of patient neutralizing antibodies (NAbs) remains 

a challenge. NAb-mediated elimination of AAV vectors has become a rate-limiting step in advancing the field 

and a determinant for repeat administration of AAV gene transfer. The fact that more than 90% of the 

population has been exposed to natural AAV2 infection, and half of those infected carry NAbs in their blood, 

highlights the significance of this problem. The study of interaction of neutralizing antibody with AAV virion 

allows us to develop effective strategies to evade NAbs activity in patients with positive NAb or for readministration. 

a. Li C, Narkbunnam N, Samulski RJ, Asokan A, Hu G, Jacobson LJ, Manco-Johnson MJ, Monahan PE; 

Joint Outcome Study Investigators. Neutralizing antibodies against adeno-associated virus examined 

prospectively in pediatric patients with hemophilia. Gene Ther. 2012 Mar;19(3):288-94

b. Li C, Diprimio N, Bowles DE, Hirsch ML, Monahan PE, Asokan A, Rabinowitz J, Agbandje-McKenna M, 

Samulski RJ. Single amino acid modification of adeno-associated virus capsid changes transduction 

and humoral immune profiles. J Virol. 2012 Aug;86(15):7752-9. 

c. Gurda BL, DiMattia MA, Miller EB, Bennett A, McKenna R, Weichert WS, Nelson CD, Chen WJ, 

Muzyczka N, Olson NH, Sinkovits RS, Chiorini JA, Zolotutkhin S, Kozyreva OG, Samulski RJ, Baker 

TS, Parrish CR, Agbandje-McKenna M. Capsid antibodies to different adeno-associated virus 

serotypes bind common regions. J Virol. 2013 Aug;87(16):9111-24. 

4. Optimization of transgene cassette: AAV is a single stranded virus; after uncoating in the nucleus, the 

single stranded AAV genome must be converted into double-stranded DNA intermediate for transcription. The 

requirement of second strand synthesis has led to the development of a new class of AAV vectors called "self 

complementary" (scAAV), which induces much higher and faster transgene expression. To further optimize 

scAAV vector for clinical trial, we have explored different elements in transgene cassettes for controlling tissue 

specific transgene expression, as well as optimization of coding and uncoding sequences that impact 

transgene expression. 

a. McCarty DM, Monahan PE, Samulski RJ. Self-complementary recombinant adeno-associated virus 

(scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther. 2001 

Aug;8(16):1248-54. 

b. Wu Z, Sun J, Zhang T, Yin C, Yin F, Van Dyke T, Samulski RJ, Monahan PE. Optimization of selfcomplementary AAV vectors for liver-directed expression results in sustained correction of hemophilia 

B at low vector dose. Mol Ther. 2008 Feb;16(2):280-9.

c. Li C, Hirsch M, Carter P, Asokan A, Zhou X, Wu Z, Samulski RJ. A small regulatory element from 

chromosome 19 enhances liver-specific gene expression. Gene Ther. 2009 Jan;16(1):43-51. 

d. Gray SJ, Foti SB, Schwartz JW, Bachaboina L, Taylor-Blake B, Coleman J, Ehlers MD, Zylka MJ, 

McCown TJ, Samulski RJ. Optimizing promoters for recombinant adeno-associated virus-mediated 

Contact PD/PI: Li, Chengwen 

Biosketches Page 19 
gene expression in the peripheral and central nervous system using self-complementary vectors. Hum 

Gene Ther. 2011 Sep;22(9):1143-53. 

5. Broad collaboration in gene therapy community: I have actively participated in studies in AAV biology 

and pre-clinical/clinical trials in the gene therapy community by collaboration or providing support, 

especially in three clinical trials including Duchenne's muscular dystrophy, canavan disease and 

hemophilia. 

a. Mendell JR, Campbell K, Rodino-Klapac L, Sahenk Z, Shilling C, Lewis S, Bowles D, Gray S, Li C, 

Galloway G, Malik V, Coley B, Clark KR, Li J, Xiao X, Samulski J, McPhee SW, Samulski RJ, Walker 

CM. Dystrophin immunity in Duchenne's muscular dystrophy. N Engl J Med. 2010 Oct 7;363(15):1429-

37.

b. Leone P, Shera D, McPhee SW, Francis JS, Kolodny EH, Bilaniuk LT, Wang DJ, Assadi M, Goldfarb O, 

Goldman HW, Freese A, Young D, During MJ, Samulski RJ, Janson CG. Long-term follow-up after 

gene therapy for canavan disease. Sci Transl Med. 2012 Dec 19;4(165):165ra163. 

c. Ishikawa K, Fish KM, Tilemann L, Rapti K, Aguero J, Santos-Gallego CG, Lee A, Karakikes I, Xie C, 

Akar FG, Shimada YJ, Gwathmey JK, Asokan A, McPhee S, Samulski J, Samulski RJ, Sigg DC, Weber 

T, Kranias EG, Hajjar RJ. Cardiac I-1c overexpression with reengineered AAV improves cardiac 

function in swine ischemic heart failure. Mol Ther. 2014 Dec;22(12):2038-45. 

d. Ling C, Wang Y, Lu Y, Wang L, Jayandharan GR, Aslanidi GV, Li B, Cheng B, Ma W, Lentz T, Ling C, 

Xiao X, Samulski RJ, Muzyczka N, Srivastava A. Enhanced transgene expression from recombinant 

single-stranded D-sequence-substituted adeno-associated virus vectors in human cell lines in vitro and 

in murine hepatocytes in vivo. J Virol. 2015 Jan 15;89(2):952-61.

Complete List of Published Work in MyBibliography: 

 

http://www.ncbi.nlm.nih.gov/myncbi/collections/bibliography/40585640 

D. Research Support 

Ongoing Research Support 

R01DK084033 Li/Samulski (MPI) 04/01/12-01/31/17 

NIH/NIDDK 

AAV Gene Therapy for AAT Deficiency 

The proposed studies test several alternative solutions including development of novel AAV virions with 

improved liver-specific targeting. 

R01 EY005951 Campochiaro P (PI), Samulski RJ(Sub PI) 05/01/2012 – 04/30/2017 

NIH/National Eye Institute – Subcontract to Johns Hopkins University 

Oxidative damage and cone cell death in RP 

The long-term objective of this collaborative effort is to evaluate protective gene therapy for the treatment and 

prevention of oxidative damage and cone cell death in human RP. 

1P01HL112761 Samulski, RJ (PI) 02/08/13-01/31/2018 

NIH/NHLBI 

NEUTRALIZING ANTIBODY & AAV FIX GENE THERAPY 

Project 1-FIX Gene Therapy and Role of AAV Nab PI 

This proposal will investigate the relationship of HLA class II phenotype with AAV neutralizing antibody against 

different serotypes, and explore the novel approach to evade neutralizing antibody activity by using Nab 

specific aptamers. 

R01 AI072176 Hirsch/Samulski (MPI) 05/15/2013 – 04/30/2018 

NIH/NIAID 

 

Contact PD/PI: Li, Chengwen 

Biosketches Page 20
	
Rational and Combinatorial Engineering of AAV Vectors 

The primary goal is to develop more efficient safe AAV vectors that overcome rate-limiting properties 

associated with current AAV vector transduction and provide a more desirable delivery reagent for future 

clinical trials. 

 

R01 AR 064369 Hirsch/Samulski (MPI) 09/17/2013 – 08/31/2018 

NIH/NIAMS 

Overcoming our clinical complications: AAV vector design for the treatment of DMD 

Explore the peptides from viruses to inhibit antigen presentation from transgene minidystrophin after AAV 

muscular delivery in canine model. 

 

Contact PD/PI: Li, Chengwen 

Biosketches Page 21
	
Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-25T09:02:19.000-04:00

PHS 398 Cover Page Supplement

OMB Number: 0925-0001

1. Project Director / Principal Investigator (PD/PI)

Prefix:

First Name*: Chengwen

Middle Name:

Last Name*: Li

Suffix:

2. Human Subjects

Clinical Trial? ❍ No ❍ Yes

Agency-Defined Phase III Clinical Trial?* ❍ No ❍ Yes

3. Permission Statement*

If this application does not result in an award, is the Government permitted to disclose the title of your proposed project, and the name,

address, telephone number and e-mail address of the official signing for the applicant organization, to organizations that may be

interested in contacting you for further information (e.g., possible collaborations, investment)?

● Yes ❍ No

4. Program Income*

Is program income anticipated during the periods for which the grant support is requested? ❍ Yes ● No

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and source(s).

Otherwise, leave this section blank.

Budget Period* Anticipated Amount ($)* Source(s)*

Contact PD/PI: Li, Chengwen 

 Page 22
	
Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-25T09:02:19.000-04:00

PHS 398 Cover Page Supplement

5. Human Embryonic Stem Cells

Does the proposed project involve human embryonic stem cells?* ● No ❍ Yes

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following

list: http://grants.nih.gov/stem_cells/registry/current.htm. Or, if a specific stem cell line cannot be referenced at this time, please check the box

indicating that one from the registry will be used:

Cell Line(s): Specific stem cell line cannot be referenced at this time. One from the registry will be used.

6. Inventions and Patents (For renewal applications only)

Inventions and Patents*: ❍ Yes ❍ No

If the answer is "Yes" then please answer the following:

Previously Reported*: ❍ Yes ❍ No

7. Change of Investigator / Change of Institution Questions

❏ Change of principal investigator / program director

Name of former principal investigator / program director:

Prefix:

First Name*:

Middle Name:

Last Name*:

Suffix:

❏ Change of Grantee Institution

Name of former institution*:

Contact PD/PI: Li, Chengwen 

 Page 23
	
Contact PD/PI: Li, Chengwen 

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-25T09:02:19.000-04:00

OMB Number: 0925-0001

PHS 398 Modular Budget

Budget Period: 1

Start Date: 04/01/2016 End Date: 03/31/2017

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A 0.00

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Organized Research_On Campus

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Darryl Mayes, 202-401-2808

Indirect Cost Rate Agreement Date 05/16/2012 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 24 
Contact PD/PI: Li, Chengwen 

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-25T09:02:19.000-04:00

PHS 398 Modular Budget

Budget Period: 2

Start Date: 04/01/2017 End Date: 03/31/2018

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A 0.00

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Organized Research_On Campus

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Darryl Mayes, 202-401-2808

Indirect Cost Rate Agreement Date 05/16/2012 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 25 
Contact PD/PI: Li, Chengwen 

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-25T09:02:19.000-04:00

PHS 398 Modular Budget

Budget Period: 3

Start Date: 04/01/2018 End Date: 03/31/2019

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A 0.00

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Organized Research_On Campus

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Darryl Mayes, 202-401-2808

Indirect Cost Rate Agreement Date 05/16/2012 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 26 
Contact PD/PI: Li, Chengwen 

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-25T09:02:19.000-04:00

PHS 398 Modular Budget

Budget Period: 4

Start Date: 04/01/2019 End Date: 03/31/2020

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A 0.00

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Organized Research_On Campus

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Darryl Mayes, 202-401-2808

Indirect Cost Rate Agreement Date 05/16/2012 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 27 
Contact PD/PI: Li, Chengwen 

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-25T09:02:19.000-04:00

PHS 398 Modular Budget

Budget Period: 5

Start Date: 04/01/2020 End Date: 03/31/2021

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A 0.00

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Organized Research_On Campus

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Darryl Mayes, 202-401-2808

Indirect Cost Rate Agreement Date 05/16/2012 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 28 
Contact PD/PI: Li, Chengwen 

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-25T09:02:19.000-04:00

PHS 398 Modular Budget

Cumulative Budget Information

1. Total Costs, Entire Project Period

Section A, Total Direct Cost less Consortium F&A for Entire Project Period ($)

Section A, Total Consortium F&A for Entire Project Period ($) 0.00

Section A, Total Direct Costs for Entire Project Period ($)

Section B, Total Indirect Costs for Entire Project Period ($)

Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period ($)

2. Budget Justifications

Personnel Justification PersonnelJustification1022072246.pdf

Consortium Justification

Additional Narrative Justification

 Page 29 
PERSONNEL JUSTIFICATION 

Chengwen Li, Ph.D., Principal Investigator, (3.6 CM Years 1-5) will have responsibility for the execution and 

completion of the proposed research and will oversee immunological analysis. In addition, he will also analyze 

experimental data and apply the results obtained towards further studies. 

Richard J. Samulski, Ph.D., Principal Investigator (0.6 CM Years 1-5) will oversee work performed by all 

personnel involved in the research proposed and be the guiding force behind all work executed. He will provide 

his expertise and knowledge in the field of AAV biology and supervise vector construction and characterization. 

Maxim Salganik, Post-doctoral Fellow, (6 CM Years 1-5) will carry out the experiments about effect of empty 

virions and AAV2 mutants as well as proteasome inhibitors on capsid antigen presentation in vitro and in vivo.

Karen Hogan, Research Specialist, (6 CM Years 1-5) will be responsible for animal experiments including 

animal care and breeding. 

Chen Xiaojing, Research Assistant, (9 CM Years 1-2, 8.4 CM Year 3, 7.8 CM Year 4, 6 CM Year 5) will be

primarily responsible for vector clone and production. She will also perform experiments to characterize AAV 

mutants isolated from humanized mice. 

Fringe benefits: Fringe benefits are requested in accordance with institutional guidelines for each position. 

Fringes for PI’s and Research Tech are calculated at for retirement and social security with Medical 

Insurance calculated at . prorated to percent effort. The Post Doc receives fringe with 

 for Medical Insurance. 

 

Contact PD/PI: Li, Chengwen 

Personnel Justification Page 30
	
 

PHS 398 Research Plan

Please attach applicable sections of the research plan, below. OMB Number: 0925-0001

Tracking Number: GRANT11947975 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-25T09:02:19.000-04:00

1. Introduction to Application

(for RESUBMISSION or REVISION only)

 Introduction1022072221.pdf

2. Specific Aims  SpecificAims1022072222.pdf

3. Research Strategy*  ResearchStrategy1022072259.pdf

4. Progress Report Publication List

Human Subjects Sections

5. Protection of Human Subjects

6. Inclusion of Women and Minorities

7. Inclusion of Children

Other Research Plan Sections

8. Vertebrate Animals  VertebrateAnimals1022072224.pdf

9. Select Agent Research  SelectAgentResearch1022072227.pdf

10. Multiple PD/PI Leadership Plan  MultiplePI_LeadershipPlan1022072225.pdf

11. Consortium/Contractual Arrangements  

12. Letters of Support

13. Resource Sharing Plan(s)  ResourceSharingPlan1022072245.pdf

Appendix (if applicable)

14. Appendix

Contact PD/PI: Li, Chengwen 

 Page 31
	
INTRODUCTION 

The current proposal is a revised grant submission based on the previous submission (R01 AI117408) entitled 

“Enhance AAV Liver Transduction with Capsid Immune Evasion”. We would like to thank the reviewers for their 

critiques and for awarding the proposal a favorable score (22 percentile). We also appreciate their 

encouraging comments that characterize the submission as “highly significant because it addresses the issue 

of antigen cross-presentation, thought to be a critical barrier to the use of AAV gene therapy for hemophilia B”,

and “the logical plan for inhibiting the CTL response to the capsid and the outstanding investigators deemed to 

be leaders in the field”. More importantly, we thank the reviewers for identifying and articulating potential 

weaknesses with respect to specific experiments and overall goals of the proposal that we could specifically 

address to improve the re-submission. We have now addressed these concerns individually below and 

accordingly made relevant changes to improve the application. Key points were identified, namely: 1) the 

approaches are not innovative and are likely to lead to only incremental progress, 2) the rationale for the 

experimental design was poorly articulated and 3) the applicants did not adequately consider the effect of preexisting immune responses on antigen presentation. 

Point 1) We agree with the reviewers about the novelty. In the resubmission application, we have combined 

Aim 1 and 2 from the previous submission and provided the new Aim 3 to isolate AAV mutants with human 

hepatocyte tropism and the ability to avoid capsid ubiquitination using innovative approaches (the combination 

of directed evolution and rational design). 

Point 2) We appreciate the concern from the reviewers. In the resubmission, we have provided more careful 

rationale for the proposal. AAV capsid antigen presentation is dose-dependent and relies on capsid 

degradation by the proteasome, and some AAV mutants or proteasome inhibitors are able to increase AAV 

transduction and block capsid antigen presentation. We hypothesize that it is feasible to develop AAV mutants 

with avoidance of AAV capsid ubiquitination to decrease AAV capsid antigen presentation and enhance 

hepatocyte transduction. 

Point 3) We acknowledge the concern from the reviewers about pre-existing immunity. Although antibody 

response can affect antigen presentation, prior studies have demonstrated that AAV antibody blocks AAV 

transduction in vitro and in vivo (e.g. no AAV genomes are detected in tissues including the liver after systemic 

administration of AAV in mice with pre-vaccination of AAV). These observations indicate that it is impossible to 

study AAV capsid antigen presentation in the setting of pre-existing antibody. Different AAV serotypes and 

mutants have been proposed to evade AAV neutralizing antibody; however, most epitopes from AAV serotypes 

and mutants are conserved. This implies that capsid specific CTL response is still able to clear AAV 

transduced cells from other serotypes or mutants. In this proposal, we propose to study the mutants with the 

ability to escape ubiquitination and degradation by the proteasome; these mutant capsids should mount no or 

low capsid antigen on the target cell surface for CTL recognition, and therefore avoid CTL mediated 

elimination. These mutants will be used to evade capsid specific CTL mediated elimination regardless of preexisting cellular immune response. 

In this submission, we have made every effort to address the minor points raised by the reviewers and to 

bolster the application with any reviewer-recommended suggestions. We thank the reviewers again for their 

constructive comments and have provided a point-by-point response to their critique below. 

1. “It is not clear how the observed data, particularly those from mutants whose transduction mechanisms have 

not been clearly understood, can be generalized or exploited to generate novel capsids that can avoid the 

CTL-mediated destruction of AAV transduced cells in Aim 3” (Reviewer 2). We overall agree with the 

reviewer’s comment; however, this is why we propose the study to investigate the effect of different mutants 

on AAV capsid antigen presentation. The majority of the concerns have been addressed in the “pitfall section”. 

2. “It is not clearly stated how the ultimate goal of the project "enhance AAV liver transduction with capsid 

immune evasion" will be achieved using the study outcomes” (Reviewer 2). In short, A, with respect to 

enhancing AAV liver transduction, we will study the effect of AAV mutants and proteasome inhibitors; B, for 

capsid evasion, we focus on AAV mutants with decreased ubiquitination and proteasome inhibitors which 

should block proteasome mediated capsid degradation. We have modified the application to emphasize their 

roles. 3. “HepG2/H2-Kb/TAP-/- xenograft model may not be the best model for this project” (Reviewer 2). We 

have deleted experiments related to the HepG2/H2-Kb/TAP-/- xenograft model in our resubmission. 4.

“Insertion of the OVA SIINFEKL peptide into the HI loop of various AAV2 and AAV8 mutants might negatively 

affect the ability to transduce cells in vitro and in vivo” (Reviewer 2). We agree with the reviewer. If the 

transduction ability is affected from these mutants with substitution of OVA peptide, we will study capsid 

antigen presentation from mutants using peptide specific CTLs induced by Ad/AAV capsid immunization even 

though the assay sensitivity is lower than OVA approach. It completely addresses the reviewer’s concern. 5.

“Biosafety consideration in the use of AAV and adenoviral vectors is not described in the application”

(Reviewer 2). We have provided biosafety consideration. 

 

Contact PD/PI: Li, Chengwen 

Introduction Page 32
	
SPECIFIC AIMS 

Adeno-associated virus (AAV) vector has been successfully applied to target the liver in clinical trials with 

hemophilia patients1,2. These trials have suggested that the AAV capsid specific cytotoxic T lymphocytes 

(CTLs) eliminate AAV vector targeted liver cells, following AAV2 or AAV8 transduction, and result in 

therapeutic failure. Our studies and others have demonstrated that both classical antigen presentation and 

cross-presentation pathways are involved in mounting an AAV capsid specific CTL response3-7. In clinical 

trials, ion exchange chromatography has been used to purify AAV vectors. Unlike CsCl purification approach, 

the chromatographic method cannot currently separate genome-containing AAV capsids (full particles) from 

empty particles. The contamination of empty virions potentially increases the AAV capsid antigen load in 

transduced cells and it has been demonstrated that empty virion contamination in vector preparations induces 

liver damage, which potentially enhances capsid antigen presentation from full virion transduction8,9. Although 

we have observed a lower capsid antigen presentation from AAV empty virion infection compared to full 

particles in vitro10, our in vivo preliminary result demonstrated that AAV empty capsids still elicit capsid antigen 

presentation. In this proposal we will investigate the kinetics of capsid antigen presentation from empty virions 

and the effect of empty particles on antigen presentation from full particle transduction (Aim 1a and 1b). We 

have demonstrated that AAV capsid cross-presentation is dependent on virion endosomal escape and 

proteasome-mediated capsid degradation in AAV transduced cells in vitro10. However, the mechanistic insights 

of the work were largely elucidated in vitro, and over a limited time period (24 to 48 hrs), and therefore it 

remains unclear which aspects of our discoveries translate in vivo regarding long-term antigen processing and 

presentation. The mechanism of capsid antigen presentation from empty virions and full particles in vivo will be 

performed using mouse models deficient in the genes responsible for classical class-I antigen presentation 

(TAP -/- mice) or classical class-II antigen presentation (Cat S -/- mice) (Aim 1c).

Our data have shown that capsid antigen presentation is dose-dependent and requires capsid ubiquitination for 

proteasome mediated degradation11,12. To decrease antigen presentation on AAV transduced cells for avoiding 

capsid specific CTL-mediated elimination, it has been proposed to modify the AAV capsid surface in order to 

enhance AAV transduction while lowering the effective dose, or to escape capsid ubiquitination13,14. It is 

unclear whether the enhancement of liver transduction with AAV mutants or a decrease in capsid ubiquitination 

influences capsid antigen presentation in vivo (Aim 2a). Proteasome inhibitors have been shown to enhance 

AAV transduction and inhibit antigen presentation12,15. However, our further in vitro study demonstrated varying 

effects of the proteasome inhibitor on capsid antigen cross-presentation in a dose related manner. A high dose 

of the proteasome inhibitor bortezomib blocks capsid antigen presentation, while a lower dose of bortezomib 

increases capsid antigen presentation without enhanced transduction. We hypothesize that proteasome 

inhibitor treatment will change the profile of AAV antigen presentation in vivo and the combination of AAV 

mutants and proteasome inhibitors will further increase AAV transduction while inhibiting capsid antigen 

presentation (Aim 2b and 2c). 

It is well-known that the transduction of AAV vectors in mouse models does not always translate into the 

human host. To address this, a mouse model xenografted with human hepatocytes has been used to develop 

AAV vectors for human liver targeting gene therapy16. In this proposal we will explore the directed evolution 

approach combined with a rational design strategy to isolate AAV vectors with human hepatocyte specific 

tropism and the ability to evade a capsid specific CTL response in humanized mice (Aim 3). Elucidation of 

AAV empty capsid antigen presentation in vivo and the development of an AAV vector with enhanced human

liver transduction and CTL immune-evasion, will allow us to design safer and more effective strategies that 

address the current clinical complications for human liver gene therapy using AAV. To address these issues, 

we will execute the following specific aims: 

1. Study the effect of AAV empty particles on AAV capsid antigen cross-presentation in vivo. 

a. The kinetics and dose-response of AAV capsid antigen presentation from AAV empty virions in vivo. 

b. The effect of empty particles on capsid antigen presentation from full-particle AAV transduction in vivo. 

c. AAV capsid antigen presentation in TAP-/- and in Cat S-/- mice. 

2. Investigate AAV capsid antigen presentation following administration of AAV mutants and/or 

proteasome inhibitors for enhanced liver transduction in vivo. 

a. Capsid antigen presentation from AAV mutants with enhanced liver transduction in mice.

b. The effect of proteasome inhibitors (high vs low dose) on natural AAV capsid antigen presentation in 

vivo. 

c. The effect of a combination of AAV mutants with proteasome inhibitors on antigen presentation in vivo. 

3. Isolate AAV chimeric capsids with human hepatocyte tropism and the capacity for CTL evasion.

a. Verify AAV human liver transduction efficiency in xenograft mice. 

b. Characterization of AAV mutants recovered from human liver xenografted mice. 

c. Investigation of capsid CTL evasion from humanized AAV mutants.

 

Contact PD/PI: Li, Chengwen 

Specific Aims Page 33
	
RESEARCH STRATEGY 

A. SIGNIFICANCE 

Adeno-associated virus (AAV) vectors have been successfully used to transduce hepatocytes in Phase I 

clinical trials in patients with hemophilia B1,2. However, clinical results have suggested that capsid specific 

cytotoxic T lymphocytes (CTLs) eliminated AAV transduced hepatocytes thus resulting in the therapeutic 

failure1,2. These observations pose an outstanding concern regarding capsid antigen presentation in AAV 

transduced cells which are recognized and eliminated by capsid specific CTLs in clinical trials. Our prior 

studies have supported classical class I antigen presentation and cross-presentation of AAV capsid epitopes3. 

Most peptides to be loaded on MHC class I molecules are generated by proteasome degradation of newly 

synthesized ubiquitinated proteins. Exogenous protein can also be presented on MHC class I molecules 

through cross-presentation. Two main intracellular pathways for cross-presentation have been described: 

endocytic (TAP, proteasome-independent) and cytosolic (TAP, proteasome-dependent) MHC class I peptide 

loading17. In the TAP-independent pathway, exogenous antigens that have been endocytosed are degraded by 

proteases, and the resulting peptides bind to MHC class I molecules in late endosomes and lysosomes. In the 

TAP dependent pathway, the internalized exogenous antigens are transferred from the endocytic pathway to 

the cytosol and degraded by proteasomes; the resulting peptides are then transported to the ER via TAP. We 

have applied pharmacological agents and demonstrated that the classic MHC class I antigen presentation 

pathway plays a major role in AAV capsid antigen cross-presentation in AAV transduced cells in vitro12. 

However, the mechanism of capsid antigen cross-presentation from AAV transduced cells in vivo is perhaps 

different from that in vitro due to the far more complex environment. 

AAV vectors purified from cesium chloride (CsCl) density gradients have been applied in clinical trials; 

however, this purification approach is not scalable. Recently, ion-exchange chromatography has been studied 

to purify AAV vectors18-21. Unlike the CsCl approach, the chromatographic method cannot separate genomecontaining particles of AAV vectors (full particles) from empty particles as it relies on the charge of the capsid 

surface. The contamination of vector preparations by empty particles inhibits transduction of genome 

containing AAV vectors and potentially increases the virus capsid antigen load in transduced cells21. Although 

empty AAV particles contain the identical protein components required for trafficking as full particles, our 

preliminary results demonstrated that capsid antigen presentation was significantly reduced in AAV transduced

cells infected with AAV2 empty virions compared to full particles in vitro12. This phenomenon may be 

interpreted as insufficient escape of these empty virions from the endosome22. However, our in vivo results 

demonstrated that capsid antigen presentation was induced from AAV empty virions. This finding is 

inconsistent with our in vitro observations and supports the likely scenario that a much more complex 

mechanism of antigen presentation occurs in vivo. Recently, empty AAV capsids were proposed to function as 

decoys in the clinical trials to allow AAV full particles to escape neutralizing antibodies23, but liver damage 

was observed following systemic administration of AAV preparations contaminated with empty particles9, 

which alludes to the possibility that empty particles enhance capsid antigen presentation. Elucidation of the 

efficiency of capsid antigen presentation from empty virions, and the effect of empty virions on capsid antigen 

presentation of full AAV capsids following in vivo administration, will address the concern of whether 

contamination of empty AAV particles is a potential risk that increases antigen presentation in AAV transduced 

liver cells in vivo (Aim 1). 

Our previous study has demonstrated that capsid antigen presentation in AAV transduced target cells is dosedependent11. To avoid capsid specific CTL mediated clearance of AAV transduced liver cells, a lower dose of 

AAV vector has been proposed to reduce the capsid antigen load in AAV transduced cells. To obtain similar 

transduction efficiencies with low vector doses, several approaches have been explored including transgene 

optimization24,25, capsid alterations26, and drug treatments to enhance transduction27-35. It has been 

demonstrated that modification of the AAV capsid can enhance liver transduction and modulate the 

immunogenicity of AAV capsid antigens13,14,36-40. Our recent study demonstrated that engraftment of the AAV9 

galactose receptor binding residues into the AAV2 virion (dual receptors) induced stronger liver transgene 

expression13. After transduction, the AAV capsid is ubiquitinated for degradation and therefore, inhibition of 

capsid ubiquitination may also enhance AAV transduction. In fact, it has been demonstrated that modification 

of capsid tyrosine residues helps AAV vectors escape ubiquitination and subsequent proteasome-mediated 

degradation, thus enhancing transduction14. The administration of these tyrosine capsid mutants results in less 

capsid specific CTL mediated elimination of AAV transduced liver cells compared to the equivalent wild type 

AAV capsid, perhaps due to faster virus trafficking to the nucleus of hepatocytes, and therefore less capsid 

antigen presentation in transduced cells41. However, it is unclear whether capsid modifications that enhance 

transduction affect capsid antigen presentation (Aim 2a). A number of pharmacological agents have also been 

used for enhancing AAV transduction, including proteasome inhibitors, DNA synthesis inhibitors and 

topoisomerase inhibitors27,28,30-34. In particular, AAV transduction with proteasome inhibitors has been tested in 

large animals32. In spite of the enhanced effect on AAV transduction, our study also demonstrated inhibition of 

 

Contact PD/PI: Li, Chengwen 

Research Strategy Page 34
	
capsid antigen presentation using a high dose of the proteasome inhibitor bortezomib12

, while increased 

antigen presentation was observed at a lower dose in vitro. It is still unclear whether proteasome inhibitor 

treatment at different doses or the combination of AAV mutants with a proteasome inhibitor affects capsid 

antigen presentation in vivo (Aim 2b and c). Investigating the effects of enhanced liver transduction or 

avoidance of ubiquitination of AAV capsid mutants and proteasome inhibitors on capsid-based antigen 

presentation will allow the community to understand the effective parameters for designing safer AAV vectors 

with enhanced liver transduction and long-term clinical efficacy. 

It has been well known that the results from mouse studies rarely translate to larger animals and humans in 

hemophilia AAV gene therapy due to species-dependent hepatocyte tropism. To develop liver targeting AAV 

virus for human clinical trials, more authentic animal models are greatly needed. Recent studies have 

demonstrated that AAV serotypes 2 and 8 could not transduce human hepatocytes efficiently in human liver 

xenografted mice42,1,2. This finding is especially relevant because AAV8 transduces mouse liver quite readily 

and with much greater efficiency than AAV2. However, the substitution of an AAV8 capsid in place of AAV2 for 

factor IX delivery in human clinical trials did not lead to a similar degree of increased factor IX expression from 

human liver, as would have been predicted by the expression advantage seen in mouse liver in vivo 1,2,43,44. 

Here, we propose to use directed evolution in combination with a rationally designed approach (mutations to 

avoid capsid ubiquitination) for selection of AAV mutants with enhanced human liver tropism and CTL evasion. 

Such enhanced reagents can be immediately transferred to clinical applications (Aim 3). The mutants 

isolated from humanized mice can also be applied in the clinical trials with pre-existing immune response but 

without neutralizing antibody (NAb). NAb blocks AAV transduction, so it is impossible to study capsid antigen 

presentation in the presence of pre-existing antibody. Different AAV serotypes and mutants have been 

proposed to evade AAV Nabs; however, most epitopes from AAV serotypes and mutants are conserved. This 

indicates that the capsid specific CTL response is still able to clear AAV transduced cells from other 

serotypes or mutants. In this proposal, we propose to develop mutants with the ability to escape ubiquitination 

and degradation by the proteasome; these mutant capsids will mount no or low capsid antigen on target cell 

surface for CTL recognition, and therefore avoid CTL mediated elimination. These mutants can be used to 

evade capsid specific CTL response regardless of pre-existing cellular immune response. 

Our labs have recently completed numerous studies that demonstrate our capability for conducting these 

investigations, including the following observations: 1) AAV capsid antigen presentation is dependent on 

proteasome-mediated capsid degradation in vitro12, 2) AAV capsid antigen cross-presentation is dosedependent in vivo11

, 3) although no capsid antigen presentation from empty virions was displayed in vitro, 

capsid antigen cross-presentation was observed in mice after systemic administration of AAV empty vectors 

(Fig. 1), 4) modification of AAV virions increases liver transduction (Fig. 5 and 6), 5) proteasome inhibitors 

enhance AAV liver transduction in vivo (Fig. 7), 6) AAV mutants have been isolated with specific cell tropism or 

the ability to cross blood-brain barrier using directed evolution approach45,46, and 7) the humanized mouse has 

been successfully established with human hepatocytes. All of these preliminary results lay the groundwork to 

develop novel AAV mutants with human hepatocyte tropism and evasion of capsid specific CTL response. The 

long-term goal is to apply this information to design safer and more effective strategies for overcoming cellbased immunity to AAV vectors, thereby generating a long-lasting therapeutic product. 

B. INNOVATION 

AAV vector gene therapy is yielding clinical data supporting the hope for the eventual treatment of many 

diseases. However, capsid specific CTL responses have been documented in patients with hemophilia B in 

several Phase I clinical trials. Over 90% of the human population is infected by AAV, and some of them have 

memory capsid CTLs in circulation47,48. AAV empty virions have been used in clinical trials as decoys for AAV 

neutralizing antibodies; however, one of the clinical concerns is that the contamination of empty particles in 

AAV vector preparation may potentially increase the AAV capsid antigen load. The detailed elucidation of AAV 

capsid antigen presentation from empty particles and the effect of empty particles on full particle capsid 

antigen presentation is innovative and timely for the guidance of future clinical trials. To decrease capsid 

specific CTL mediated killing of AAV transduced target cells, it has been proposed to use lower doses of more 

potent AAV vectors, due to the dose-dependent nature of capsid antigen presentation11. To achieve this goal, 

capsid mutants and proteasome inhibitors have been explored to enhance AAV transduction and also to 

interfere with capsid antigen presentation. Elucidation of the effect of AAV mutants or proteasome inhibitors 

that enhance AAV transduction and decrease AAV capsid antigen presentation is both important and 

innovative. To explore effective approaches of successfully evading capsid CTL response for human clinical 

trial, the development of AAV mutants by rational design and directed evolution in the human hepatocyte 

xenografted mouse is novel and straightforward. 

C. APPROACH 

The primary objectives of the proposal are the following: (i) to study the antigen presentation from AAV empty 

particles in vivo, (ii) to investigate the effect of AAV mutants and proteasome inhibitors that enhance liver 

 

Contact PD/PI: Li, Chengwen 

Research Strategy Page 35
	
Fig. 2. The kinetics of capsid antigen presentation after AAV8OVA transduction in mice. 

Particles of AAVOVA/AAT virus (1×1011) were injected intravenously into C57BL/6 mice, and at the 

indicated time points, 5×106 CFSE-labeled OT-1 T cells were transferred. Ten days after transfer, 

proliferation of CD8+ OT-1 T cells was measured by flow cytometry. (A) Average T cell proliferation 

and standard deviation for four mice. (B) Average proliferation index (PI) and standard deviation. 

**p<0.01, *p<0.05 compared with control mice without AAV treatment.

Fig. 1. Capsid antigen presentation after 

systemic administration of AAV2 empty 

virions. A. Representative flow cytometric 

histograms. B. Average T cell proliferation 

and standard deviation for four mice. 

transduction on capsid antigen cross presentation, (iii) to isolate AAV mutants with human hepatocyte tropism 

and the capacity to avoid capsid antigen presentation. 

C1. Study the effect of AAV empty particles on AAV capsid antigen cross-presentation in vivo. 

Rationale. Ion-exchange chromatography has been used to purify AAV vectors at clinical quantities; however, 

this approach is unable to eliminate empty particle contamination as it relies on capsid charge and not overall 

particle density49. The empty particles inhibit AAV transduction and potentially increase the capsid antigen load 

in transduced cells21,23. A recent study demonstrates that the empty virions in AAV vector preparations induce 

liver damage, which may up-regulate antigen presentation following 

AAV transduction9. These observations pose an outstanding 

concern in the clinical trials using AAV vector preparations 

contaminated with empty particles. Our previous study in the 

HepG2 cell line demonstrated that far less antigen presentation was 

induced from empty virions compared to full particles

50

. However, 

the antigen presentation mechanism from AAV empty capsid in vivo

is likely much more complicated than that in cell lines considering 

the shorter experimental time period in vitro and more 

environmental factors influencing antigen presentation in vivo. Thus, 

the studies from cell lines in vitro may not represent all the 

alternative processes of capsid antigen that can take place over 

extended periods of time in vivo. A recent study demonstrated that 

empty AAV8 virions do not induce strong antigen presentation in 

mice when compared to full particles51; in this study the marker 

antigen, OVA peptide SIINFEKL, was located at the N-terminal of 

VP2. These viruses were assembled from two AAV helper 

plasmids: one to encode AAV VP1 and VP3, the other one for VP2 

with the OVA peptide insertion at N-terminal. The virus preparation 

may be composed of VP1/VP2/VP3 (intact virus), VP2/VP3, or VP3 only. The latter two viruses do not have 

infectivity since they cannot escape from endosome due to no PLA2 domain. Additionally it is unknown 

whether VP1 and VP2 are cleaved in the endosome after their exposure on the virion surface. In our 

preliminary studies, using AAV virions with OVA peptide integration in VP3, we found that AAV2-OVA empty 

virions induced comparable capsid antigen presentation to full particles after systemic administration (Fig. 1), 

which is inconsistent to our in vitro observation that much lower capsid antigen presentation is mounted from 

empty virions compared to full particles. 

It has been shown that transgene expression gradually increases following AAV2 administration in mice, peaks 

at week 6, then remains stable for long-term24. To address whether the kinetics of antigen presentation of 

capsid epitopes in vivo

corresponds with the 

dynamics of transgene 

expression, we injected 

1x1011 particles of

AAV2-OVA/AAT 

intravenously (IV). At 

various time points 

post-injection, splenic 

OT-1 T cells labeled 

with CFSE dye were 

administered to the 

treated mice. Ten days 

post-transfer, OT-1 T 

cell division was 

measured by flow 

cytometry. As shown in 

Fig. 2, over days 3-12 

and days 21-30, OT-1 T cell division was significantly increased in AAV2-OVA/AAT vector-treated animals 

versus control recipients. However, no difference was observed for OT-1 cell proliferation between AAV2-OVA 

and the control groups over days 41-50 and days 61-70 (p>0.05). This result indicates that antigen crosspresentation from the AAV2 capsid occurs early after AAV2 systemic administration11. Similar kinetics and 

efficiency of antigen presentation was also observed from an AAV8-OVA vector after systemic administration. 

 

Contact PD/PI: Li, Chengwen 

Research Strategy Page 36
	
17.5%

Control 

A BCells in division (%)

40%

5e10

18.3%

1e10

17.8%

1e9

18.5%

1e8

87.8%

2e11

**

*

C

**

FIG. 3. Capsid antigen presentation after AAVOVA 

transduction is dose responsive in vivo. Various doses of 

AAV2OVA/AAT vector were injected intravenously into 

C57BL/6 mice and 3 days later, CFSE-labeled OT-1 T cells 

were transferred. On day 10 after transfer, OT-1 T cell 

proliferation in the spleen was assessed via flow cytometry. 

(A) Representative flow cytometric histograms. (B) Average T 

cell proliferation and standard deviation of four mice. (C)

Average proliferation index (PI) and standard deviation. 

**p<0.01, *p<0.05 compared with control mice without AAV 

treatment.

Fig. 4. Mechanism of capsid antigen cross-presentation in 

HepG2/H-2Kb cells. (A) Effect of inhibitors on antigen 

presentation. (B) Antigen presentation by transduced AAV2-

OVA, 75HD/AN, and VP3-only virus. (C) Effect of AAV NLSs on 

capsid antigen presentation. *P < 0.05 and **P < 0.01 when 

compared with AAV2-OVA. 

To investigate the dose response of capsid antigen presentation in vivo, varying particle numbers of the AAV2-

OVA vector were injected into mice to examine OT-1 T cell stimulation. When compared to the no treatment 

control, a dose of 5x1010 AAV2-OVA particles induced the proliferation of OT-1 T cells (Fig. 3). Importantly, 

there was no proliferation of spleen cells in groups 

with doses of AAV2-OVA less than or equal to 1x1010

particles. Together, these results demonstrate that 

efficient T cell stimulation occurs only at high doses of 

AAV vectors11. In this specific aim, we will further 

investigate the kinetics and dose-response of AAV (2 

and 8) empty capsid antigen cross-presentation 

following systemic application of AAV empty virions in 

a mouse model (C1.1). Due to the mixture of full 

particles and empty virions in the vector preparations, 

we will also study the effect of their interactions on 

capsid antigen presentation (C1.2).

Effective AAV transduction is composed of several 

steps, including vector binding to the cell surface via a

receptor-mediated mechanism followed by 

endocytosis through clathrin-coated pits. The particles 

then travel from early endosomes to late endosomes 

followed by escape to the lysosome or peri-nuclear 

localization. Once inside the nucleus, viral uncoating 

and the initiation of second-strand synthesis is 

proposed to occur, followed by transgene 

expression52-55. To address the mechanism of capsid 

antigen presentation after AAV transduction, we 

incubated HepG2/H2-kb cells with different chemicals

at non-toxic concentrations for 1 hr before AAV2-OVA 

virions were added. After 24 hr, the spleen cells from 

OT-1 mice were co-cultured with AAV2-OVA transduced HepG2/H2-kb cells overnight. Then, activated OT-1 

spleen cells were detected by flow cytometry for CD8 and CD69 staining, an early marker of T cell early 

activation. Consistent to prior work, both ammonium 

chloride and chloroquine (inhibitiors of acidification

in the endosomes) inhibited AAV2 transduction52. 

Compared to antigen presentation from AAV2-OVA 

transduction without treatment, over 90% inhibition 

of antigen presentation was observed in groups 

treated with ammonium chloride and chloroquine 

(Fig. 4A). To analyze the participation of proteases 

in the processing of AAV capsids after transduction, 

two different protease inhibitors, leupeptin H and 

pepstatin A, were tested. Incubation of HepG2/H2-

kb cells with the AAV2-OVA vector in the presence 

of leupeptin or pepstatin partially blocked OVA 

presentation (Fig. 4A), suggesting that OVA antigen 

processing from the AAV2-OVA virion required the 

participation of some proteases12. Two proteasome 

inhibitors, MG132 and Bortezomib, as with many 

previous studies, enhanced AAV2 transduction. 

However, OVA antigen presentation was completely 

diminished in the presence of these inhibitors (Fig. 

4A). This result indicates that AAV2-OVA virions, or 

derived peptides, translocate to the cytosol of 

HepG2/H2-kb cells and are degraded by the 

proteasomes12. We also demonstrated that Brefeldin 

A (BFA) significantly suppressed AAV2 transduction, and inhibited capsid antigen presentation (Fig. 4A). This 

strongly suggests that AAV2-OVA derived peptides are transported to cell surface through the Golgi complex 

after translocation to ER12. Collectively from the above study with pharmacological agents, it is concluded that 

 

Contact PD/PI: Li, Chengwen 

Research Strategy Page 37
	
capsid antigen presentation is dependent on proteasome-mediated degradation of the AAV capsid, although 

the acidic environment in endosomes is required for effective AAV vector escape.

The N termini of VP1 and VP2 in the AAV capsid are necessary for AAV vector subcellular trafficking. Two 

functional motifs have been identified: a phospholipase dominant (PLA2) domain and putative nuclear 

localization signals (NLSs) BR1, BR2, and BR322,56-59. The AAV PLA2 region plays a role in disrupting the 

membrane of the vesicular compartment and allows the virus to escape into the cytosol from the 

endosome/lysosomes. It has been demonstrated that a mutant with a two residue substitution of PLA2

(AAV2/75HD/AN) is unaffected in regards to cell surface binding and virus endocytosis, but greatly attenuates 

PLA2 activity thereby decreasing vector transduction. To further confirm the role of AAV endosome/lysosome 

escape in capsid antigen presentation, we incubated AAV2-OVA/75HD/AN vectors with HepG2/H2-kb cells for 

capsid antigen presentation analysis. As shown in Fig. 4B, compared to the AAV2-OVA vector, significant 

reduction of antigen presentation was observed with the AAV2-OVA/75HD/AN mutant vector (Fig. 4B). Our 

previous study demonstrated that VP3 only virus, which does not contain the PLA2 domain on its N-terminus, is 

not able to enter the nucleus and fails to transduce AAV2 permissive cells30. Similar to the AAV2-

OVA/75HD/AN mutant, infection with AAV2-OVA VP3 only virus led to a reduction of antigen presentation in 

HepG2/H2-kb cells (Fig. 4B)

12

. We have demonstrated that mutations of three potential NLSs (BR1, BR2 and 

BR3) dramatically decreased transduction, due to defective nuclear entry of the mutant particles. To 

understand the relationship of AAV capsid antigen presentation in relation to the cytoplasmic localization of 

uncoated capsids, we infected HepG2/H2-kb cells with these mutants with OVA peptide substitution and 

examined antigen presentation on the cell surface. The BR1 mutant vector infection led to significantly 

decreased antigen presentation (Fig. 4C). Interestingly, both BR2 and BR3 mutants induced similar antigen 

presentation when compared to the AAV2-OVA vector (Fig. 4C). These results suggest that uncoating in the 

nucleus is not required for AAV capsid antigen presentation after infection12. Due to toxicity of chemical agents 

and inconsistency of antigen presentation from empty virion infection in vitro and in mice, it is impossible to 

study the mechanism of capsid antigen cross-antigen presentation in vivo using these chemicals and mutants. 

Therefore, we will study the mechanism of antigen presentation from empty capsids using our animal models 

deficient in genes responsible for classic class-I antigen presentation (TAP -/- mice) or class-II antigen 

presentation (Cat S -/- mice) in Aim1.3. 

C1.1. The kinetics and dose-response of AAV capsid antigen presentation from AAV empty virions in 

vivo. AAV empty and full particles from AAV2-OVA and AAV8-OVA will be administered to mice at different 

doses. At different time points post-injection, CFSE-labeled spleen cells from OT-1 mice will be transfused into 

treated mice. The proliferation of OT-1 CD8 cells will be analyzed as a measure of OVA epitope presentation.

Empty AAV production. AAV empty virions are produced by transfection of 293 cells with the AAV-OVA helper 

and Ad helper plasmids without AAV transgene cassette plasmid, and then purified by CsCl gradient 

centrifugation. The titer of the produced empty particles will be determined using a Western blot with 

antibodies specifically detecting intact virions (A20 and ADK8 antibodies for AAV2 and AAV8, respectively). 

Animal experiments for kinetics of antigen presentation from AAV empty capsids. Three groups of experiments 

will be designed: AAV empty capsids, AAV full particles and vehicle -treatment (PBS). After AAV administration 

(1x1011 particles) to C57BL mice via tail vein injection, at different time points (days 3, 21, 41 and 61 post AAV 

administration), 5x106 CFSE-labeled OT-1 spleen cells will be transfused into these treated mice via IV 

administration. Ten days after the OT-1 cell infusion, mice will be sacrificed and the proliferation of OT-1 CD8 

cells will be analyzed. Animal experiments for dose-response of AAV capsid antigen presentation from AAV 

empty capsids. Escalating doses of AAV particles (108, 109, 1010, 1011 and 1012) will be injected into C57BL 

mice via tail vein. Three days later, CFSE-labeled OT-1 cells will be infused for an OT-1 CD8 proliferation 

assay11. Three groups of mice will be assigned: AAV empty, AAV full particle and vehicle treatment. Statistics 

analysis: Data are expressed as means ± standard error. A Student T-test (StatView, SAS institute, NC) will be 

used to determine significant differences between the different treatment groups. 

C1.2. The effect of empty particles on AAV capsid antigen cross presentation from full particle AAV 

transduction in vivo. First, we will study the effect of AAV empty capsids on transgene production from AAV 

full virion transduction in vivo with different ratios of empty to full particles. Then, the effect of empty capsids on 

capsid antigen presentation from AAV full capsids will be explored. Also we will study the effect of full particles 

on capsid antigen presentation from empty particles in vivo. 

Animal experiment for transgene production. 1x1011 particles of AAV/AAT spiked with different doses of AAV 

empty capsids (0, 1x1010, 3x1010, 1x1011, 3x1011, 1x1012) will be administered to C57BL mice via tail vein 

injection. At weeks 1, 2, 4, 6 post-injection, the AAT concentration in the blood will be measured. Animal 

experiment for capsid antigen presentation. To determine the effect of empty capsids on capsid antigen 

presentation from full virions, 1x1011 particles of AAV-OVA/AAT (2 and 8) spiked with different dose of AAV or 

AAV-OVA empty particles (0, 1x1010, 3x1010,1x1011, 3x1011, 1x1012) will be administered into C57BL mice via 

tail vein injection. At 3 days post-injection, CFSE labeled OT-1 spleen cells will be transfused into mice for an 

 

Contact PD/PI: Li, Chengwen 

Research Strategy Page 38
	
in vivo OT-1 CD8 cell proliferative analysis. To examine the effect of full particles on capsid antigen 

presentation from empty virions, 1x1011 particles of AAV-OVA empty virions spiked with different dose of 

AAV/AAT or AAV-OVA/AAT viruses (0, 1x1010, 3x1010,1x1011, 3x1011, 1x1012) will be administered to C57BL 

mice via tail vein injection for a capsid specific CTL proliferation assay as described above. 

C1.3. The mechanism of empty capsid antigen presentation in vivo. Transporter associated with Antigen 

Processing (TAP) is a member of the ATP-binding-cassette transporter family. It delivers cytosolic peptides 

into the ER for binding to nascent MHC class I molecules. Cathepsins are proteases located in the endosomes 

or lysosomes. Cathepsin S (Cat S) may participate in the degradation of exogenous antigenic proteins to 

peptides for presentation on MHC molecules. In this sub-Aim, we will use mice with TAP or Cat S deficiency to 

determine which pathway (endocytic or cytosolic) is involved in capsid antigen presentation in AAV transduced 

liver cells from full particles or empty virions in vivo. TAP-/- mice and Cat S -/- mice, as well as wild type C57BL 

controls, will receive AAV-OVA vector via tail vein injection. Transgene production will be measured using an 

ELISA for AAT. Antigen presentation following AAV-OVA administration will be analyzed via spleen T cell 

proliferation from OT-1 mice. 

Transgene expression analysis. 1x1011 particles of AAV/AAT will be injected into mice (TAP-/-, Cat S-/- and 

C57BL) via the tail vein. AAT levels in blood will be determined by ELISA3. In vivo T cell proliferation assay.

1x1011 particles of AAV-OVA vectors will be injected into mice (TAP-/-, Cat S-/- and C57BL) via the tail vein. 

Three days later, 5x106 CFSE labeled T cells from the spleen of OT-1 mice will be transferred via IV injection. 

Ten days later, T cells from the spleen and the liver are harvested and stained with antibodies specific for CD8. 

The frequency of proliferating T cells will be determined12. 

Anticipated results, potential pitfalls and alternative approaches: We expect that capsid antigen 

presentation from empty virions is dose-dependent and occurs early, similar to full particles of AAV in vivo. It is 

anticipated that antigen presentation from empty particles is relatively long after administration due to impaired 

nuclear trafficking compared to full virions. High levels of empty particle contamination are expected to inhibit 

AAV transgene production from the co-administered full particles. It is likely that empty particles will enhance 

antigen presentation from full particles or vice versa. Our preliminary data demonstrated that empty virions are 

deficient in endosomal escape and travel to the nucleus in cultured cells 30. Therefore, it is possible that both 

endocytic and cytosolic MHC class I antigen presentation pathways are involved in capsid antigen presentation 

from empty AAV infection in vivo, which suggests that comparable or lower capsid antigen presentation from 

empty virions will be observed when compared to full particles in TAP -/- mice. If so, we will perform 

experiments to track empty particle trafficking in the liver using fluorescence Cy5 labeled virions to explore the 

difference of behavior between full and empty virions60,61. Although chemical inhibition of proteases can inhibit 

AAV capsid antigen presentation, the capsid antigen presentation from AAV transduction may not be impacted 

from the absence of the single protease, cathepsin S, in deficient mice. It is possible that effective AAV capsid 

antigen presentation is induced in TAP-/- mice since a TAP independent and proteasome dependent pathway 

for cross presentation has been identified62. If this is the case, we will study whether proteasome-mediated 

degradation of AAV virions plays a major role in capsid cross-presentation after AAV administration in vivo

using proteasome inhibitors such as bortezomib or carflizomib. Considering the toxicity of proteasome 

inhibitors, the drugs’ short half-life in mice (rapidly cleared following intravenous administration with the peak at 

about 30 minutes)63,64, and the potential enhancement of antigen presentation observed when using low doses 

of such drugs (see Fig. 8), we will use the highest non-lethal dose of proteasome inhibitor with repeated drug 

delivery (three days) and analyze the antigen presentation over a very short time period after capsid specific 

CD8 cell infusion. One other concern is that TAP-/- mice may have lower overall MHC class expression; 

however, it has been shown that strong antigen presentation from endogenous or exogenous protein is 

nonetheless observed in these mice65. In general, the approaches proposed in this aim are straightforward and 

we have all the required reagents and extensive experience elucidating the CTL response to AAV capsids. As 

we perform these techniques on a regular basis, we don’t foresee any problems in carrying out these 

experiments. 

C2. Investigate effect of AAV mutants and proteasome inhibitors on AAV capsid antigen presentation. 

Rationale. Our preliminary data indicate that AAV capsid antigen presentation is dose-dependent, and that a 

lower dose of AAV vectors reduces the overall antigen presentation (Fig. 3). Therefore, it is reasonable to 

explore effective strategies to achieve the necessary therapeutic levels while using lower vector doses. To 

achieve this goal, several approaches have been exploited to enhance AAV liver transduction including 

utilization of different serotypes, genetic modification of AAV capsids, and optimization of AAV vector 

cassettes, as well as the application of chemical reagents. However, it is unclear how these strategies 

(especially for AAV mutants and pharmacological agents) impact AAV capsid antigen cross-presentation in 

target cells while having enhanced liver transduction. 

AAV viruses exploit heparan sulfate (HS), galactose (Gal), or sialic acids (Sia) as primary receptors for cell 

surface binding66,67. Different AAV strains also require subsequent interaction with co-receptors for cellular 

 

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Research Strategy Page 39
	
AAV2 AAV2D AAV2D/585E AAV8

Luc expression (RLU)

A

B

Fig. 6. Transgene expression from AAV2 

mutants. A. representative images of vector 

induced luciferase activity. B. Fold increase 

of luciferase activity using AAV mutant 

capsids compared to AAV2.

AAV2 2G9 2i8 2i8G9 AAV9

Fig. 5. Transgene expression from AAV2 

mutants with modification of primary 

receptors. BALB/c mice were administered 

AAV vectors at a dose of 1 × 1011 particles via 

the tail vein, and bioluminescent images were 

collected at day 18 post-injection. 

uptake66,67. Recently, key amino acid residues involved in Gal recognition by AAV9 capsids were identified68. 

Modification of receptor binding sites on the AAV capsid can either change transgene profile or transduction 

efficiency. We have pioneered rationale design studies of AAV 

capsids, for instance mutating the critical residues for primary 

receptor binding site between different serotypes and 

demonstrating that the AAV2/AAV8 chimera AAV2i8 displayed an 

altered transduction profile. AAV2i8 selectively transduces cardiac 

and whole-body skeletal muscles with high efficiency and loses 

liver tropism38. Further studies integrating AAV9 primary receptor 

Gal binding residues into the AAV2 capsid (AAV2G9) found that 

AAV2G9 has dual receptor function and exploits Gal and heparan 

sulfate receptors for infection. Of particular interest, AAV2G9 

retains a similar tropism to AAV2 but confers more rapid onset and 

higher liver transgene expression in mice. Similarly, engraftment of 

the Gal footprint onto the AAV2i8 (AAV2i8G9) also induced higher 

transduction in muscles and liver, comparable with AAV913 (Fig. 

5). In addition, we have demonstrated that the modifications at the 265 residue of the AAV2 capsid change 

AAV2 tissue tropism and the immune profile69. Insertion of an aspartic acid at the 265 residue of the AAV2 

capsid (AAV2D) induced much higher muscle transduction than AAV269. Similarly, systemic administration of 

AAV2D also induced higher liver transduction than AAV2 (Fig. 6). It is well known that residue 585 Arg 

contributes to the AAV2 heparin binding capacity and that mutation of the AAV2 heparin binding site 

(AAV2/585E) ablates AAV2 liver tropism36. However, insertion of Asp at residue 265 of AAV2/585E capsid 

increased the liver tropism to a similar transduction efficiency as observed using AAV8 (Fig. 6). These mutants 

(AAV2G9, AAV2D and AAV2D/585E) can be used at lower doses to achieve similar transgene gene 

expression in transduced liver to AAV2. 

In addition to investigating AAV mutants with enhanced liver transduction, the AAV virion surface has also 

been modified to avoid ubiquitination for capsid degradation14,39,40. 

Ubiquitination is a post-translational modification of a substrate 

protein that can direct degradation via the proteasome. The process 

of ubiquitination initiates with ubiquitin binding to lysine residues on 

the protein substrate or to the amino group of the protein's Nterminus70,71. It has been shown that the mutants with surfaceexposed tyrosine altered to phenylalanine reduce AAV capsid 

ubiquitination by elimination of potential capsid phosphorylation. 

Additionally, the triple mutant (AAV2 Y-F) with modification of three 

tyrosine residues (Y730+500+444F) induces 3 fold higher liver 

transduction14. However, decreased transgene expression with the 

triple mutant was still observed over time in the presence of capsid 

specific CTLs, which indicates that mutant capsid antigens are still 

able to be displayed on the surface of transduced liver cells for 

capsid specific CTL recognition, albeit at a low efficiency of 

presentation41. To reduce AAV capsid ubiquitination, some other 

studies have been performed following mutation of the lysine 

residues on the AAV capsid39,40. The mutants AAV2 K532R (lysine at 

residue 532 of AAV2 capsid was mutated to arginine) and AAV8 

K137R demonstrated lower ubiquitination than AAV2 and AAV839,40, 

respectively, and both AAV mutants showed higher liver transduction 

in mice although a similar liver transduction efficiency from AAV8 

K137R and AAV8 was observed from several groups including 

ours72. In spite of the decreased capsid ubiquitination for mutants 

with Y-F or K-R, further detailed studies are definitely needed to confirm the efficiency of antigen presentation 

from these mutants in vivo. In this study, we hypothesize that the mutants with multiple different modifications 

(AAV2G9, AAV2D, AAV2 Y-F and AAV2 K532R for AAV2, AAV8 Y-F and AAV8 K137R for AAV8) will further 

enhance liver transduction and decrease capsid antigen presentation (Aim 2.1).

A number of chemical reagents have been used for enhancing AAV transduction, including proteasome 

inhibitors such as MG-132 and bortezomib, DNA synthesis inhibitors such as hydroxyurea (HU) and 

aphidicolin, and topoisomerase inhibitors such as etoposide and camptothecin27,28,30-34. Thus far, the leading 

candidate for enhancing rAAV transduction in vivo is the proteasome inhibitor bortezomib, which has been 

demonstrated to increase transgene expression 3- to 6-fold in a large-animal study32. We have further 

 

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Research Strategy Page 40
	
-500

-400

-300

-200

-100

0

100

200

1nM 10nM 100nM 1uM 10uM

Antigen presentation

Inhibition %

0

200000

400000

600000

800000

Luc expression (lu)

A

B

Fig. 8. The effect of bortezomib on capsid 

antigen presentation. A. Luciferase transgene 

expression. B. capsid antigen presentation. The 

x-axis indicates the concentration of bortezomib.

Fig. 7. The effect of proteasome inhibitors 

on AAV2 transduction. Balb/C mice 

received 1x1011 particles of AAV2/luc and 

0.5 mg bortezomib/kg or 1 mg carfilzomib/kg 

at the same time. Transduction was assayed 

by live imaging at 7 days post AAV injection. 

identified another proteasome inhibitor, carfilzomib, which also augments AAV liver transduction (Fig. 7). As 

bortezomib enhances AAV transduction the greatest, we also studied the effect of this proteasome inhibitor on 

AAV capsid antigen presentation, and found that a high 

concentration of bortezomib inhibits capsid antigen presentation with 

enhanced transgene expression (Figs. 4 and 7). In contrast, a lower

concentration of bortezomib (10nM) increased antigen presentation 

from AAV2-OVA transduced HepG2 cells without increasing 

transgene expression (Fig. 8), and an intermediate dose (100 nM) 

enhanced both transduction and antigen presentation. This in vitro

result brings up the concern of whether the utilization of proteasome 

inhibitors to enhance AAV transduction may impart reduced, or 

perhaps enhanced, AAV capsid antigen presentation in patients. To 

address the effect of proteasome inhibitors on capsid antigen 

presentation, we will study AAV capsid antigen cross-presentation in vivo using proteasome inhibitors 

bortezomib and carifilzomib (Aim 2.2). 

It is assumed that the mutants with several different individual modifications induce much higher liver 

transduction than mutants with a single modification, and the combination of proteasome inhibitor with AAV 

mutants will further enhance AAV liver transduction. Finally, we will explore the effect of the combination of 

proteasome inhibitors with AAV mutants described above on capsid antigen presentation in vitro and in mice

(Aim 2.3).

C2.1. Study capsid antigen cross-presentation from AAV mutants. 

First, we will clone OVA SIINFEKL peptide into the HI loop of AAV2 capsid mutants (AAV2G9, AAV2D, AAV2 

Y-F and AAV2 K532R) or AAV8 mutants (AAV8 Y-F and AAV8 K137R) to generate the constructs pXR2G9-

OVA, pXR2D-OVA, pXR2 Y-F-OVA, pXR2 K532R-OVA, pXR8 Y-F-OVA and pXR8 K137R-OVA. Next, we will 

make mutants with multiple mutations, then clone for the AAV2 mutant (defined as AAV2-MT4) which includes 

4 different modifications: AAV2G9, AAV2D, AAV2 Y-F and AAV2 

K532R, and for the AAV8 mutant (AAV8-MT2) which is 

composed of 2 modifications: AAV8 Y-F and AAV8 K137R, will 

be generated. These constructs will be used to package the AAT 

or luciferase transgene. After virus production, mutant vector 

characterization will be performed in vitro and in vivo. Then, 

these viruses will be used to examine the AAV capsid antigen 

presentation in HepG2/H2kb. Finally, AAV mutants will be 

injected into C57BL mice via the tail vein for a capsid antigen 

presentation analysis. Dose-response and kinetics of capsid 

antigen presentation will be determined. 

Clone of AAV mutants. Site-directed Mutagenesis and PCR will 

be used to generate AAV capsid mutants with single mutations. 

Ubiquitin conjugation assay and immunoblotting. A ubiquitination 

assay of viral capsids will be performed with a ubiquitin–protein 

conjugation kit (Boston Biochem, Cambridge, MA). Briefly, after 

conjugation fraction A, conjugation fraction B and ubiquitin are 

mixed with energy buffer; the conjugation reaction is then 

initiated by adding 3×108heat-denatured AAV viral particles and 

incubated at 37°C for 4 hr. Equal volumes of sodium dodecyl 

sulfate (SDS)-denatured ubiquitinated samples are then resolved 

on a 4–20% gradient gel. The ubiquitination pattern for the 

various viral particles is detected by immunoblotting of the 

samples with mouse anti-ubiquitin monoclonal antibody (P4D1) 

and horseradish peroxidase (HRP)-conjugated anti-mouse IgG1 (Cell Signaling Technology, Boston, MA). 

VP1, VP2, and VP3 capsid proteins are detected with AAV clone B1 antibody (Fitzgerald, North Acton, MA). 

Luciferase imaging in mice. Imaging will be performed with a Xenogen IVIS Lumina system after IP injection of 

the D-luciferin substrate. Bioluminescence imaging analysis is carried out with Living Image software 

(PerkinElmer, Waltham, MA). Antigen presentation in mice: C57BL mice will receive AAV mutant vectors 

(1x1011 particles) via tail vein injection. Antigen presentation will be analyzed for capsid specific T cell 

proliferation at different time points (day 3, 21, 41, 61) after AAV administration to examine the kinetics of 

antigen presentation. In the second experiment, different doses of AAV mutants (109, 1010, 1011 and 2x1011

particles) will be administered into C57BL mice via tail vein injection, and 3 days after AAV injection, OT-1 

spleen cells will be infused for CD8 T cell proliferation analysis as described above. 

 

Contact PD/PI: Li, Chengwen 

Research Strategy Page 41
	
C2.2. The effect of proteasome inhibitors on AAV capsid antigen presentation in vivo. Proteasome 

inhibitors (bortezomib and carfilzomib) at different doses (10ug, 100ug, 1mg and 10mg/kg) will be coadministered to mice with AAV2-OVA/luc or AAV8-OVA/luc via tail vein injection, and transgene expression 

and antigen presentation (efficiency and kinetics) will be detected via live imaging of luciferase activity. 

Parameters for toxicities including body weight and blood chemistry will be monitored by the UNC animal 

clinical chemistry core facility (https://www.med.unc.edu/anclinic). 

Antigen presentation in mice. C57BL mice will be treated with proteasome inhibitors at the above doses with 

co-administration of 1x1011 particles of AAV-OVA vector via tail vein injection. Antigen presentation will be 

analyzed for capsid specific T cell proliferation at different time points (day 3, 21, 41, 61) after AAV 

administration. 

C2.3. The effect of proteasome inhibitors on antigen presentation in vivo using liver enhanced AAV 

mutants. After systemic administration of AAV mutants (AAV2-OVA-T4 or AAV8-OVA-T2) and proteasome 

inhibitors in mice (C1.1 and C1.2), the transgene expression and antigen presentation will be examined. 

Mouse experiments: we will administer the proteasome inhibitors with AAV2-OVA-MT4 or AAV8-OVA-MT2 into 

C57BL mice, and transgene expression will be examined to compare with AAV2-OVA and AAV8-OVA, 

respectively. For the antigen presentation assay, different doses (108, 109, 1010, 1011) of AAV2-OVA-MT4 and 

AAV8-OVA-MT2 mutants will be studied in the presence of proteasome inhibitors. 

Anticipated results, potential pitfalls and alternative approaches: We have demonstrated that substitution 

of the HI loop in AAV2 and AAV8 virions with the immunodomain OVA SIINFEKL peptide does not change 

AAV infectivity or tissue tropism11,73. We expect that HI loop swapping with the OVA peptide in AAV2 or AAV8 

mutants will neither influence transduction efficiency or tropism in vitro or in mice. Although higher transgene 

expression is expected, it is also anticipated that AAV mutants with Y-F or K-R change will induce lower capsid 

antigen presentation than wtAAV-OVA in HepG2, or similar capsid antigen presentation from other AAV 

mutants since our preliminary results suggest that uncoating of AAV virions in the cytoplasm plays a major role 

in AAV capsid antigen presentation12. Consistent with expected results in HepG2 cells, AAV2-OVA or AAV8-

OVA mutants with Y-F or K-R modification will induce higher transgene expression than wtAAV-OVA after tail 

vein injection with lower antigen presentation. Despite the anticipated differences, it is possible that similar 

capsid antigen presentation will be induced between these mutants and wtAAV-OVA. Similar to wtAAV2-OVA 

and wtAAV8-OVA, stronger capsid antigen presentation is expected to occur early after AAV2 or AAV8 mutant 

administration and will gradually decrease over time. It is expected that the kinetics of capsid antigen 

presentation from AAV mutants’ transduction is shorter than that from wtAAV2-OVA and wtAAV8-OVA. Our 

preliminary results have shown that the proteasome inhibitor bortezomib at a high dose has dual function: 

increased AAV transduction and decreased antigen presentation. However, when a relatively low dose is used, 

AAV transduction is increased or not changed, but antigen presentation is increased (Fig. 8). It is anticipated 

that the proteasome inhibitor carfilzomib will have a similar profile to bortezomib in affecting transgene 

expression and antigen presentation in vitro. In vivo, both proteasome inhibitors are hypothesized to decrease 

capsid antigen presentation at high-doses after AAV-OVA transduction, while antigen presentation will 

increase when low doses are administered, consistent with in vitro observations (Fig. 8). It is possible that high 

doses of proteasome inhibitors will also induce high capsid antigen presentation, perhaps because the antigen 

presentation assay requires a longer time in vivo and the effect of proteasome inhibition is short. When the 

AAV2-MT4 mutant which contains 4 different individual modifications (AAV2G9, AAV2D, AAV2 Y-F and AAV2 

K532R) or AAV8-MT2 mutant which contains two modifications (AAV8 Y-F and AAV8 K137R) is administered 

in combination with proteasome inhibitors in mice, transgene expression is expected to be much higher than 

that from a single individual mutant or proteasome inhibitor alone. However, the capsid antigen presentation 

from the AAV2-MT4 or AAV8-MT2 mutant will be undetectable or much lower than that from the mutant alone 

or wt AAV2/AAV8 in the presence of proteasome inhibitors in mice. It is possible that the AAV2-MT4 mutant 

may not induce higher transgene expression than the single mutants. If this is the case, we will test the 

mutants with the combination of two or three individual modifications. The other concern is that the insertion of 

the OVA SIINFEKL peptide into the HI loop of various AAV2 and AAV8 mutants might negatively affect the 

transduction ability and tropism. If this is the case, we will study capsid antigen presentation from mutants 

using wild type capsid specific CTLs generated from mutant capsid immunized mice although this approach 

sensitivity is lower than that with OVA substitution. Several approaches will be used to generate wild type 

capsid specific CTLs in mice including: Ad vector delivery of mutant capsid, peptide pulsed dendritic cell 

immunization, or direct immunization with peptides since several peptides from the AAV capsid (AAV2 VP1 

aa702-710, AAV8 VP1 aa517-525) have been identified as CD8 epitopes in C57BL mice6. While it is 

impossible to describe every potential outcome that we may encounter, we have strong preliminary data to 

support our experimental design. We also have extensive experience in adapting our protocols to unexpected 

outcomes. 

C3. Isolate AAV mutants with human hepatocyte tropism and immune-evasion capacity. 

 

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Fig. 9. Fluorescence micrographs of green 

fluorescent protein (GFP) transgene expression in 

CS1 cells transduced with AAV serotypes 1–9 

(except 7) and the novel variant chimeric-1829 at a 

multiplicity of infection (MOI) of 1,000 for 48 hours.

Fig. 10. Engraftment of human liver in FRG 

mice. FRG mice received 1x106frozen fetal or 

fresh adult liver cells. The human albumin in 

the blood was measured by ELISA at 12 

weeks and 5 weeks after xenograft. The data 

represent the average of 2 mice and SD.

Rationale. Numerous studies have demonstrated that AAV8 is the most efficient and specific vector to 

transduce mouse liver among the known serotypes. However, the relative efficiency of AAV8 transduction in 

the liver of the mouse was not observed in primates and dogs when the same vector dose/kilogram body 

weight was used; much lower liver transduction was achieved in these larger animals than in mice44,74. This 

observation suggests that the results generated in murine tissues may not translate well into large animals or 

humans. It is imperative to develop AAV mutants with human hepatocyte tropism and ability to evade 

proteasome mediated degradation of capsid. Recently, a mouse model with a xenografted human liver has 

been used to study AAV vector transduction in human liver cells and to develop human liver-tropic AAV 

mutants42. However, the information is still lacking for a transduction profile of human hepatocytes from AAV 

serotypes and mutants which have demonstrated high mouse liver tropism. To address this concern, we will 

used a xenografted mouse model with human liver cells to examine the transduction efficiency of AAV 

serotypes and mutants developed from Aim 2 in human liver 

cells and elucidate whether AAV mutants with immune evasion 

and mouse liver tropism from Aim 2.1 also have higher 

transgene expression in human hepatocytes (C3.1). 

DNA shuffling is a powerful process for directed evolution, 

which generates diversity by recombination, combining useful 

mutations from individual genes. Single and multigene traits 

that require many mutations for improved phenotypes can be 

evolved rapidly. We have utilized this approach to generate an 

AAV DNA shuffling library (from AAV capsid serotypes 1-6, 8 

and 9) and isolated a cos-1 integrin minus specific variant 

(AAV1829) that had enhanced transduction in cos-1 cells 

compared to all parent serotypes (Fig. 9)

45

. Using the same 

approach, after IV administration of the AAV capsid library, we 

were able to isolate two mutants from neuron rich cells in rats 

with limbic seizure activity induced by kainic acid. These 

mutants transduced the cells that were localized to the piriform 

cortex and ventral hippocampus after IV administration, 

indicating that novel AAV vectors have been created that can 

selectively cross the seizure-compromised BBB and transduce 

cells46. Our in vitro studies have demonstrated that AAV capsid 

degradation via the proteasome plays a major role for effective 

capsid antigen presentation, and it is well-known that lysine residues are direct targets for host cell 

ubiquitination and therefore mutating them can reduce protein ubiquitination and subsequent proteasomemediated degradation. Mutations of capsid lysine or tyrosine residues have led to low capsid ubiquitination to 

evade capsid specific CTL mediated elimination of AAV transduced cells41. From the above observations, we 

will apply the strategy of combining directed evolution with the AAV capsid library including rationally designed 

capsids with ubiquitination avoidance, to explore human liver-specific AAV capsids with the ability to avoid 

capsid CTLs in a xenograft mouse model (C3.2 and C3.3). 

C3.1. Verify AAV human liver transduction efficiency in 

xenograft mice. To study AAV transduction in human 

hepatocytes, we will first establish a xenografted mouse model with 

human liver cells. AAV serotypes and the mutants developed in 

Aim 2 will be tested in this mouse model. Besides nude mice, 

some other mouse strains have been explored to establish human 

liver xenografts including immuno-deficient Rag-/-/Ilrg2r-/- (NRG) 

mice with u-PA (Alb-uPA+/+), NRG mice with active Caspase 8 

fused with FK506-binding domain (AFC8) and NRG mice with 

fumarylacetoacetate hydrolase deficiency (FRG)75-78. Compared to 

other strains, FRG mice have demonstrated high efficiency of 

human liver cell repopulation, no limitation with regard to age of the 

xenograft and easy breeding77. In this proposal, we will use FRG 

mice (from YecurisTM, Portland, OR) to perform human liver 

xenografts. In FRG mice, we have successfully grafted human liver 

cells from frozen fetal liver and fresh adult liver (Fig. 10). 

Establishment of human liver xenografted mouse model.

Hepatocytes isolated from normal human livers will be purchased 

from Triangle Research Labs. Following the Yecuris protocol, at 24 

 

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Research Strategy Page 43
	
hrs prior to transplant, 1.25X109 pfu/25g of Ad/uPA virus will be administered to each mouse by retro-orbital 

injection, and drinking water containing SMX/TMP, but no nitisinone will be provided. 1X106 hepatocytes in 100 

ml will be injected into the inferior pole of the spleen. Animal body weights and survival rates will be closely 

monitored. The concentration of human albumin will be detected by ELISA using a commercial kit at months 2, 

6, 12, 18 to determine the engraftment efficiency of human hepatocytes. AAV transduction in human liver cells 

in xenografted mice. Since self-complementary (sc) AAV induces much higher transduction than conventional 

single-stranded AAV vectors, scAAV/GFP from 12 serotypes and 8 mutants (AAV2G9, AAV2D, AAV2 Y-F, 

AAV2 K532R, AAV8 Y-F, AAV8 K137R, AAV2-MT4, AAV8-MT2) from Aim 2 will be administered to 

xenografted mice via systemic application at the dose of 1x1011 particles (5x1012/kg). At week 6 after AAV 

injection, mice will be perfused and fixed, and the liver will be separated and sectioned. The antibodies against 

human albumin will be used to identify human cells using a PE-labeled secondary antibody. Double positive 

liver cells, GFP+ and PE+, will be observed using fluorescent microscopy and indicate human cell 

transduction.

C3.2. Selection of AAV mutants in human liver xenografted mice. High-throughput screening/ selection of 

capsid mutants from AAV libraries has been carried out earlier and appears to be a promising strategy to 

develop human liver specific AAV chimerics with the ability to evade capsid specific CTL mediated elimination 

of transduced cells. Several studies, including ours, have provided a solid rationale for carrying out directed 

evolution in vivo in xenografted mice46,79,80. First, we will rationally mutate AAV capsids from serotypes and 

mutants from Aim 2 which will be used for construction of the AAV capsid shuffling library based on the 

prediction of ubiquitination sites. Several available software programs will be used for this purpose including 

UbiPred, Composition of k-Spaced Amino Acid Pairs, and Prediction of Ubiquitination Sites with Bayesian 

Discriminant Method. Systematical analysis of scores from different programs will be performed for choosing 

top amino acids (lysine-K, serine-S and threonine-T) for mutation (K→R, S/T→A). Mutated AAV capsids will be 

digested with DNase, the digested capsid DNA fragments will be assembled into AAV capsid plasmid library, 

which will be used to generate an AAV capsid library. After injection of this AAV library into human liver 

xenografted mice in the presence of human adenovirus, human hepatocytes from mouse liver will be isolated 

using cell sorting to then isolate human hepatocyte tropic AAV mutants. 

DNA shuffling and the generation of a new AAV capsid library. The techniques outlined herein were developed 

by our lab46,80,81. Briefly, lysine and serine/threonine in AAV capsids from serotypes 1-12 and 8 mutants 

(AAV2G9, AAV2D, AAV2 Y-F, AAV2 K532R, AAV8 Y-F, AAV8 K137R, AAV2-MT4, AAV8-MT2) from Aim 2 

will be mutated into arginine and alanine, respectively, using random mutagenesis based on software 

predication and preliminary data from Aim 3.1. DNA shuffling will be carried out using a collection of capsid 

genes representing 12 different AAV serotypes and 8 mutants with lysine or serine/threonine modification. 

Following limited DNase digestion, the capsid fragments from these AAV serotypes will be assembled, 

amplified, and cloned into a wtAAV backbone to generate a shuffled AAV capsid plasmid library. This plasmid 

library then will be used to produce a shuffled AAV vector library that will be used for in vivo selection of livertropic AAV variants having the capacity to evade capsid specific CTL response. Directed evolution in human 

liver xenografted mice. The AAV vector library (1x1011 particles) will be injected into human liver xenografted 

mice with adenovirus Ad5 dl309 (1x1012 particles) via the tail vein. After 48 hrs, liver lysates will be generated 

by freeze-thaw cycles, heated to 56oC for 30 min to inactivate adenovirus, and then clarified by centrifugation 

to remove cellular debris. The pooled supernatant will be injected IV, and this process is then repeated for 

three more cycles. After each cycle, low-molecular DNA (Hirt DNA) will be extracted from liver to evaluate the 

replication yield by qPCR using AAV rep gene-specific primers. Following this iterative selection, capsid DNA 

sequences from isolated liver DNA will be amplified through PCR and cloned into the pXR shuttle vector82

. 

Individual clones, each potentially representing a chimeric capsid DNA sequence, will be sequenced and 

recombinant vectors packaged with the GFP will be generated for in vitro and in vivo characterization. 

Additionally, structural analysis of these mutants will be performed (as detailed below). Isolation of human liver 

cells. Mice will be perfused with cold PBS followed by collagenase to isolate liver cells. The liver cells labeled 

with FITC conjugated mouse anti-human HLA-ABC antibody will be sorted using flow cytometry. In vivo

characterization of AAV mutants in xenografted mice. 1x1011 particles of 10 recovered mutant viruses encoding 

GFP, and the best serotype for human liver transduction based on result from C3.1), will be injected (IV) into 

xenografted mice to determine the transduction in human liver cells. Structural analysis. AAV mutants will be 

subjected to structural characterization by molecular modeling studies using Pymol® and SWISS-MODEL®. 

PDB files of the VP3 subunit of different AAV serotypes are now available and models can be generated as 

needed using other AAV strains as templates45,79. Sequence alignment analysis using Vector NTI® will carried 

out to determine which sequences are conserved in wild type AAV serotypes. Mapping of different domains 

onto the capsid surface will be carried out by generating 3D ribbon models of dimers, trimers and pentamers 

that represent the two-fold, three-fold, and five-fold axes of symmetry, which can be generated readily on the 

 

Contact PD/PI: Li, Chengwen 

Research Strategy Page 44
	
viper database (viperdb.scripps.edu). Molecular modeling facilities are available to us through the Structural 

Bioinformatics facility at UNC-CH. 

C3.3. Investigation of immune-evasion from humanized AAV mutants. Mutants isolated from humanized 

mice will be further characterized for their immune evasion ability including ubiquitination capacity in vitro, and

for antigen presentation in AAV mutant -transduced cells in vitro and in vivo. For antigen presentation, the HI 

loop of 2 mutants with high human liver tropism from C3.2 will be substituted for the OVA SIINFEKL peptide. 

In vitro antigen presentation is performed in HepG2/H2kb cells and in vivo analysis in C57 mice. 

Detection of AAV capsid ubiquitination in AAV mutant transduced cells. 2x106 cells, mock-treated or treated 

with MG132, are infected with AAV mutants isolated from humanized mouse liver at 5x103 particles per cell for 

4 h at 37°C. Cells are harvested and lysed in 0.8 ml of radioimmunoprecipitation (RIPA) buffer. Cell lysates are 

precleared with 10 μl of protein G PLUS-agarose (Santa Cruz Biotechnology) and then are incubated with 10 

μl of mouse anti-AAV monoclonal antibody clone B1at 4°C for 1 h, followed by the addition of 30 μl of protein G 

PLUS-agarose. After overnight incubation at 4°C, the beads are washed four times with 1 ml of ice-cold RIPA 

buffer and resolved by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE). After 

transfer to a nitrocellulose membrane, the blot is probed with mouse anti-ubiquitin monoclonal antibody P4D1, 

followed by horseradish peroxidase-conjugated secondary antibody. After the final washes, the ubiquitinated 

viral protein is visualized with an enhanced chemiluminescence system (Amersham Pharmacia). Antigen 

presentation in mice: C57BL mice will receive AAV mutant vectors (1x1011 particles) or AAV2OVA via tail vein 

injection. Antigen presentation will be analyzed at different time points (day 3, 21, 41, 61) to examine the 

kinetics of antigen presentation. In the second experiment, different doses of AAV mutants (109, 1010, 1011 and 

2x1011 particles) will be administered into C57BL mice via tail vein injection, and 3 days later, OT-1 spleen cells 

will be infused to examine the dose-response of antigen presentation. 

Anticipated result, potential pitfalls and alternative approaches: Synthesis of AAV capsid libraries has 

already been successfully achieved by our group. In addition, we have demonstrated the ability to isolate novel 

AAV mutants with the ability to cross the blood-brain barrier using an in vivo screening approach. We are 

confident that we will be able to extend these technical skills to obtain novel chimeric AAV capsids by directed 

evolution following injection of the AAV library in human liver xenografted mice. We do not anticipate any 

problems with harvesting organs and subsequent analysis of clones derived from multiple rounds of cycling. 

We anticipate that AAV mutants can be obtained in human liver xenografted mice. These mutants are 

expected to have decreased ability to bind ubiquitin and mount lower capsid antigen presentation, and also 

will be pre-disposed towards efficient human liver transduction. We carried out numerous studies using these 

protocols and have extensive experience; therefore, we do not have technical limitations in performing these 

experiments. Alternatively, we will isolate liver cells after AAV injection and amplify the AAV variants in the 

presence of Ad in vitro. It is also possible that the yield of AAV virus recovered from a xenografted liver will be 

too low for subsequent amplification. In this case, an intermediate step will be taken in which AAV genomes 

containing Hirt DNA prepared from the liver will be co-transfected into 293 cells with the helper plasmids pXR2 

and pXX6-80. The chimeric AAV particles will then be used to infect 293 cells with wtAd, and the resulting 

variant AAV vector library will be injected into mice. As another alternative, variant capsid genes will be PCR 

amplified from Hirt DNA prepared from liver. The resulting library of amplicons will be subcloned into a wtAAV 

vector backbone, which will be used to produce variant AAV virus. It is possible that our strategy will not 

generate AAV capsids with improved transduction efficiencies compared with existing AAV capsids (e.g. 

AAV3). As an alternative approach to isolate AAV mutants with human hepatocyte tropism and the ability to 

escape capsid ubiquitination, we will make AAV shuffled library from 12 AAV serotypes and different mutants 

without any further substitutions of lysine and serine or threonine. This library will be injected into humanized 

mice for isolation of AAV mutants with human liver tropism. The isolated mutants will further modified at the 

residues of lysine, serine or threonine to decrease ubiquitination ability. However, the mutants derived from 

further modifications after selection may decrease its human hepatocyte tropism. The same concern as in Aim 

2 is that the OVA peptide substitution in mutants might affect the transduction ability. Nevertheless, we believe 

that our approach proposed in this aim will at a minimum lead to the development of several AAV mutants that 

induce low capsid antigen presentation and have human hepatocyte tropism– a significant outcome. 

Summary: The final objectives of this proposal are to explore novel AAV mutants with human hepatocyte 

tropism and the ability of avoiding proteasome mediated AAV capsid degradation. We anticipate that the 

overall proposed research will have a major impact on AAV vector development, and on the consideration of 

rational design of vectors. We feel our preliminary data are strong and very supportive of the experimental 

design presented in our 3 aims. We are well positioned with reagents and animal models to rigorously test 

these hypotheses. In short, AAV vector biology and the host immune response have now taken center stage 

following Phase I clinical studies. We hope to engage in pre-clinical studies that will better clarify this concern 

and the potential to explore effective approaches that may deflect future challenges of CTL responses to AAV 

transduced cells in vivo. 

 

Contact PD/PI: Li, Chengwen 

Research Strategy Page 45
	
VERTEBRATE ANIMALS 

Protocols for these projects are pending at the UNC-CH Institutional Animal Care and Use Committee and will 

be submitted using Just in time procedures. The Institutional Animal Care and Use Committee (IACUC) will 

review and approve the proposed use of animals before the activity is initiated. Details of the animal 

experiments can be found in the Research Design and Methods section. Regarding the use of vertebrate 

animals we will address the five points as required by the NIH instructions: 

1) These studies will require the use of mice. The total number of 3490 mice is required for experiments 

proposed in this application. The experiments proposed will utilize mice with TAP deficiency, Cathepsin S 

deficiency, wild type C57BL mice, OT-1 mice and NOD-Scid-IL2Rγnull (Nod-Rag1null-IL2rγnull, NSG) with 

fumarylacetoacetate hydrolase deficiency (FRG) mice. 

Aim 1. Study the effect of AAV empty particles on AAV capsid antigen cross-presentation. 

Aim 1.1. The kinetics and dose-response of AAV capsid antigen presentation from AAV2 empty 

virions in vivo. 

60 C57BL mice are required for kinetics = 5 mice/group x 3 groups (empty, full and no treatment) x 4 

time points 

90 C57BL mice are needed for dose-response = 5 mice/group x 3 groups x 6 doses (0, 108, 109, 1010, 

1011, 1012) 

60 OT-1 are required for spleen cells. 

For Aim 2.1, the total number of mice is 60 + 90 + 60 =210 including 150 C57BL mice and 60 OT-1 mice. 

Aim 1.2. The effect of empty particles on AAV capsid antigen cross presentation from full-particle 

AAV transduction in vivo. 

6 groups (0, 1x1010, 3x1010,1x1011, 3x1011, 1x1012 empty or full spiked with 1x1011 full or empty particles 

AAV2/AAT, respectively) and C57 mice will used. 

For effect of empty on transgene expression from AAV full virions, 5 mice x 6 groups = 30 

For effect of empty on capsid antigen presentation from AAV full virions, 5 mice/group x 6 groups x 4 

time points = 120 

For effect of full virion on capsid antigen presentation from empty, 5 mice/group x 6 groups x 4 time 

points = 120 

100 OT-1 mice are needed for spleen cells 

For aim 2.2, the total number is 30 + 120 + 120 + 100 = 370 including 270 C57BL mice and 100 OT-1 

mice. 

Aim 1.3. AAV capsid antigen presentation in TAP-/- and in Cat S-/- mice.

For antigen presentation, 5 mice/group x 3 strains (TAP-/-, Cat S-/- and C57BL) x 2 serotypes (AAV2 and 

8) x 2 (vector and control) x 2 (empty and full virion) = 120

20 OT-1 mice for OVA specific T cells. 

For Aim 1.3, the total mice number is 120 + 20 = 140 including 40 TAP-/- mice, 40 Cat S -/- mice, 40 

C57BL mice and 20 OT-1 mice. 

For Aim 1, the total mice number is 210 + 370 + 140 = 720. 

Aim 2. Investigate AAV capsid antigen presentation following administration of AAV mutants or 

proteasome inhibitors for enhanced AAV liver transduction. 

Aim 2.1. Capsid antigen presentation from AAV mutants with enhanced liver transduction in mice. 

For transgene expression: 95 C57BL mice are needed: 5 mice/group x 19 viruses (AAV2, AAV9, 

AAV2G9, AAV2G9-OVA, AAV2D, AAV2D-OVA, AAV2 Y-F, AAV2 Y-F-OVA, AAV2 K532R, AAV2 

K532R-OVA, AAV8, AAV8 Y-F, AAV8 Y-F-OVA, AAV8 K137R, AAV8 K137R-OVA, AAV2-MT4, AAV2-

MT4-OVA, AAV8-MT2, AAV8-MT2-OVA).

 

Contact PD/PI: Li, Chengwen 

Vertebrate Animals Page 46
	
For in vitro antigen presentation, 30 OT-1 mice are needed. 

 For dose response, 300 mice are needed: 6 (109, 3x109, 1x1010, 3x1010, 1x1011 particles of AAV vector 

and control) x 10 viruses (AAV2OVA, AAV2G9-OVA, AAV2D-OVA, AAV2 Y-F-OVA, AAV2 K532R-OVA, 

AAV8OVA, AAV8 Y-F-OVA, AAV8 K137R-OVA, AAV2-MT4-OVA, AAV8-MT2-OVA) x 5 mice/group. 

For kinetics study, 400 mice are required: 4 time points (day3, day21, day 41 and day 61) x 2 (each 

mutant + control) x 10 viruses (AAV2OVA, AAV2G9-OVA, AAV2D-OVA, AAV2 Y-F-OVA, AAV2 K532ROVA, AAV8OVA, AAV8 Y-F-OVA, AAV8 K137R-OVA, AAV2-MT4-OVA, AAV8-MT2-OVA) x 5 

mice/group. 

100 OT-1 mice are needed. 

For Aim 2.1, the total mice number is 95 + 30 + 300 + 400 + 100 = 925 including 795 C57BL mice and 

130 OT-1 mice 

Aim 2.2. The effect of proteasome inhibitors on AAV capsid antigen presentation in vivo. 

 For transgene expression from single drug treatment, 100 C57BL mice are needed: 5 doses (including 

control without treatment) x 5 mice/group x 2 proteasome inhibitors (bortezomib and carfilzomib) x 2 

serotypes (AAV2 and AAV8). 

 For antigen presentation from single drug treatment, 640 C57BL mice are needed: 4 time points (day3, 

day21, day 41 and day 61) x 2 (each dose of drug and control) x 4 dose (Table 1) x 5 mice/group x 2 

proteasome inhibitors x 2 serotypes (AAV2 and AAV8). 

200 OT-1 mice are needed to provided capsid specific CTLs. 

For Aim 2.2, the total mice number is 100 + 640 + 200 = 940 including 740 C57BL mice and 200 OT-1 

mice. 

 Aim 2.3. The effect of combination of AAV mutants with proteasome inhibitors on antigen 

presentation in vivo. 

For dose response, 300 mice are needed: 5 (108, 109, 1010, 1x1011 particles of AAV vector and control) x 

4 viruses (AAV2OVA, AAV2-MT4-OVA, AAV8OVA, AAV8-MT2-OVA) x 5 mice/group x 3 (2 proteasome 

inhibitors and control). 

60 OT-1 mice are needed. 

For aim 2.3, the total number is 300 + 60 = 360 including 300 C57BL mice and 60 OT-1 mice. 

For aim 2, the total number is 925 + 940 + 360 = 2325 

Aim 3. Isolate AAV mutants with human hepatocyte tropism and immune-evasion capacity. 

Aim 3.1. Verify AAV human liver transduction efficiency in xenograft mice.

 For examination of human hepatocyte transduction efficiency from AAV serotypes and mutants, 100 

mice are needed: 5 mice/AAV vector x 20 AAV vectors(12 serotypes, and 8 mutants AAV2G9, AAV2D, 

AAV2 Y-F, AAV2 K532R, AAV8 Y-F, AAV8 K137R, AAV2-MT4, AAV8-MT2). 

For aim 3.1, the total number is 100 FRG mice. 

 

Aim 3.2. Selection of AAV mutants in human liver xenografted mice.

 For selection of AAV mutants with human hepatocyte tropism, 20 FRG mice will be required for isolation 

of AAV mutants: 4 cycles of amplification x 5 mice/cycle. 10 mutants will be tested in human liver 

xenograft mice. So 55 mice are needed: 5 mice/group x 11 (10 mutants isolated from humanized mouse 

and best serotype or mutant from Aim 3.1). 

For aim 3.2, the total number is 20 + 55 = 75 FRG mice. 

Aim 3.3. Investigation of immune-evasion from humanized AAV mutants 

 For investigation of antigen presentation from AAV mutants isolated from humanized mouse, 2 mutants 

with the best human hepatocyte transduction will be chosen for antigen presentation analysis in vitro and 

in vivo. For dose response, 90 mice are needed: 6 (109, 3x109, 1x1010, 3x1010, 1x1011 particles of AAV 

vector and control) x 3 viruses (2 mutants and AAV2OVA) x 5 mice/group. 

 

Contact PD/PI: Li, Chengwen 

Vertebrate Animals Page 47
	
 For kinetics study, 120 mice are required: 4 time points (day3, day21, day 41 and day 61) x 2 (each 

mutant + control) x 3 viruses (2 mutants and AAV2OVA) x 5 mice/group. 

 60 OT-1 mice are needed. 

 For aim 3.3, the total number is 90 + 120 + 60 = 270 including 210 C57BL mice and 60 OT-1 mice. 

For aim 3, the total number is 100 + 75 + 270 = 445 

The total number of mice required for this proposal is 720 (Aim 1) + 2325 (Am 2) + 445 (Aim 3) = 3490. 

 

2) The use of animal models in the arena of gene therapy is essential for vector development and for 

evaluation of safety about immune response to capsid and efficacy of planned protocols. It is clear that no 

human clinical trial can be considered prior to vector evaluation and its proven success in animal models. 

Employing animal model is essential for the ability to accurately predict the outcome of a gene therapy 

protocol. The mice with TAP deficiency, cathepsin S deficiency in this study are chosen since these mice are 

deficient in gene products which involve in antigen presentation and results from study in these mice will help 

us understand the mechanism of AAV capsid antigen presentation and explore the novel approaches to inhibit 

capsid specific CTL mediated elimination of AAV transduced target cells in vivo. OT-1 mice are chosen due to 

transgenic of specific TCRs to H-2b OVA epitopes. C57BL mouse has H-2kb haplotype and presents OVA 

SIINFEKL peptide complex with H-2kb molecules on the cell surface, which can be recognized by OT-1 spleen 

cells and stimulates OT-1 CD8 cell proliferation. FRG mice has been successfully used to establish humanized 

model with liver due to deficiency of immune system and repopulation potential of xenografted liver cells in 

FRG mice. We will use FRG mice to engraft human liver to establish human liver xenograft mouse model. 

3) The mice will be housed at the Department of Laboratory Animal Medicine (DLAM) facility located at the 

Genetic Medicine building on the UNC-CH campus. All animals are cared for by experienced technicians and 

veterinarians with special training in the handling of each of the aforementioned animal species. 

4) All studies will be conducted in a manner to minimize all discomfort, distress, pain and injury. Animals that 

develop significant discomfort due to the procedures will be EUTHANIZED. 

5) The methods of euthanizing animals will be consistent with the recommendations of the panel of Euthanasia 

of the American Veterinary Medical Association. All mice will be euthanized by CO2 asphyxiation. 

 

Contact PD/PI: Li, Chengwen 

Vertebrate Animals Page 48
	
BIOHAZARDS 

Adeno-associated virus (AAV) is known not to cause any diseases in human or animals, so AAV vector is noninfectious and not hazardous materials. 

Adenovirus can cause respiratory illness and various other illnesses such as gastroenteritis, conjunctivitis, 

cystitis and rash. The production of adenovirus and experiments with adenovirus (mice experiments) will be 

performed in Biosafety Level 2. 

 

Contact PD/PI: Li, Chengwen 

Select Agent Research Page 49
	
MULTIPLE PI LEADERSHIP PLAN 

The Li and Samulski groups have been collaborating with one another for a number of years, bringing together 

expertise in the fields of immune response and AAV vector development for gene therapy applications, 

respectively. The new R01 further exploits this unique relationship to characterize a topic that has clinical 

significance, namely enhanced AAV liver transduction and decreased capsid antigen cross-presentation. 

Drs. Li and Samulski will both provide oversight for the proposed work. These 2 investigators will be 

responsible for the implementation of the leadership plan, carrying out the specific aims, and ensuring that the 

work is in compliance with DHHS and NIH policies including those for biosafety and animal research. 

Specifically, Dr. Li will oversee experiments related to capsid antigen presentation. Dr. Samulski will be 

responsible for AAV mutant deveopment. Dr. Li will serve as contact PI and assume responsibility for: i) fiscal 

and administrative management, and ii) maintaining communication between the 2 groups through monthly 

meetings. Notably, the 2 groups are highly interactive and are both located within the UNC Gene Therapy 

Center in Chapel Hill, NC. Dr. Li will be responsible for communication with the NIH and submission of annual 

reports jointly prepared with Dr. Samulski. Publication authorship will be based on the relative scientific 

contributions of the PIs and key personnel.

 

Contact PD/PI: Li, Chengwen 

Multiple PI Leadership Plan Page 50
	
 

Contact PD/PI: Li, Chengwen 

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RESOURCE SHARING PLAN 

The PI of the application will implement the reagent and data sharing plan. We anticipate that several types of 

reagents will be developed that will be of broad interest to the gene transfer community nation-wide. The DNA 

sequences for mutated capsid and data from patients that appear in articles will be made available through the 

UNC Vector Core via the internet or through the National Center for Biotechnology Information (NCBI), 

National Library of Medicine (NLM), NIH, national resource for molecular biology information. We anticipate 

that deposition of such data would be done at the time of publication, but early deposition, e.g., at the time of 

manuscript acceptance, will be encouraged. Published reviews and manuscripts will be posted with a link to 

the PubMed library (when available) on our website www.med.unc.edu/genether and will submit electronic 

copies of final peer-reviewed manuscripts, upon acceptance of publication, the National Library of Medicine’s 

PubMed Central (PMC). The searchable PMC archive will provide greater public access and permanent 

preservation of NIH-supported research, as well as provide free, full-test publications on the internet as stated 

in the NIH policy to promote immediate public access to research findings (Feb, 2005). 

VIRAL VECTORS: With respect to new viral vectors, the UNC Vector Core has a standing policy regarding the 

mechanism of notification and distribution of new vector reagents for individuals in the research community at 

large. This policy includes the following components: 

a. After new (AAV and Chimeric) viral vectors have been developed, the Core will assume the responsibility of 

maintaining the parental seed lot and will produce large quantities for the investigator’s needs.

b. For individuals who may be interested in these reagents, we will place the description of the reagent on the 

Vector Core Website www.genetherapy.unc.edu/jvl.htm. Distribution to outside investigators will require written 

permission form the PI responsible for generating the viral vector. 

 

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Resource Sharing Plans Page 55