summary of changes
untitled h2

A Phase I Study of Stem Cell Gene Therapy for HIV Mediated by Lentivector Transduced, Pre-Selected CD34+ Cells Version 9.0 NCI Protocol \#: AMC-097 Local Protocol \#: AMC-097 NCI Version Date: 07FEB2022 Protocol Date: 07FEB2022  
I. Recommendations **from Approval with Recommendations Memorandum for Protocol**  
Version 8.0 (dated 10SEP2021) from R. Little, dated 24SEP2020:  
| Version 8.0 (dated 10SEP2021) from R. Little, dated 24SEP2020:  # Section Comments 1. Appendix VII What is being measured by ELISA for Monitoring for immune response  to TRIM5α? 2.5.4.1 PI Response: This procedure was designed to be used if any immune  response was being developed against the gene modified cells. We  hypothesized that we would initially infer that an immune response  toward the cells was occurring if the levels of in vivo gene marking  decreased and eventually went to zero over time post-infusion. To  date, none of the infused patients have displayed gene marking which  would suggest an immune response toward the cells is occurring.  Therefore, we have not needed to perform this assay.  2. Appendix VII Please expand on as to how the team plan to Monitor of changes in the  HIV reservoir and viral diversity in PBMCs?  2.5.4.3, B. PI Response: As described in Section 2.5.4.3, B in order to  characterize changes in the HIV reservoir and viral diversity blood  samples will be collected at baseline, 2 and 10 weeks and then 6, 12-  and 24-months post-transplant for HIV reservoir characterization.  These studies will include quantitative PCR analysis of PBMCassociated HIV DNA, RNA (including unspliced and multiply spliced  forms) and 2-LTR circles. In addition, single genome sequencing of  HIV gp120 and HIV pol will be performed to understand sequence  evolution following transplantation and detection of minority  resistance variants. Single-copy HIV from plasma will also be  determined at each of these time points. Samples will be sent to the  Timothy Henrich Laboratory at University of California, San  Francisco.AMC 097 (Version 9.0) 07FEB2022 i   |
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AMC 097 (Version 9.0) 07FEB2022 i  
NCI Version Date 07FEB2022  
CLIENT CONFIDENTIAL  
| II.                             | Scientific and Substantive Changes:                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
|---------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| #                               | Section                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Description of Change                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 3.                              | Protocol  Schema                                                                                                                                                                                                                                                                                                                                                                                                                                                             | The ART withdrawal period has been extended from 12 weeks to until at  one of the ART resumption criteria are met. This will allow sufficient  time for the viral load to spike and then decrease and provided more data  for evaluating effects of the transformed cells.                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| 2.4 5.1                         |                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| 4.                              | 2.4                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Monitoring before and during the ART withdrawal period has been  updated to include required COVID-19 testing prior to ATI (and as  needed) and weekly testing for CD4/CD8 counts, viral load, and clinical  assessments (review of medical and medication history, height, weight,  BSA, neurological exam, and toxicity assessment) to closely monitor for  acute retroviral syndrome and opportunistic infections (OI). Clinical  assessments may be performed remotely to minimize the study burden on  participants.                                                                                                                                                                                                 |
| 2.4.2 7.5                       |                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| App - 1C                        |                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| Appendix-XI Appendix XII        | In addition, several appendices have been added including an ATI  disclosure form for participants, ATI risk mitigation counseling forms,  and PrEP information sheets for participants. The ATI disclosure form must be reviewed with participants prior to ATI. ATI risk mitigation  counseling must be performed prior to-ATI and weekly during ATI, and  may be performed remotely. PrEP information sheets should be given to  participants as a resource prior to ATI. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| Appendix XIII                   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| 5.                              | 2.4.1                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | ART resumption criteria have been revised to expand on the clinical and viral load safety parameters. The viral load criterion has been revised to  require ART to be reinstated if there are persistent increases in HIV viral  load over a period of time rather than relying on an extremely high single  value. Participants with an initial viral load greater than 400 copies/ml  during weekly testing must be retested within 3 business days or as soon  as possible. Clinical criteria have been expanded to require ART  resumption in the event of non-infectious symptoms suggestive of severe  acute retroviral syndrome for consistency with clinical care standards and  other HIV cure-intent protocols. |
| 2.4.2                           |                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| App - 1C                        |                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| 6.                              | 2.4.3                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Post ART withdrawal monitoring has been added. Participants will have  CD4/CD8+T cell counts, HIV viral load, and clinical assessments at 4, 8,  and 24 weeks after restarting ART to ensure that viral load and CD4  counts remain at a safe level. If viral load is not below the level of  quantification by Week 8, participants will be evaluated every four weeks  until the viral load is confirmed to be less than the level of quantification. In addition, Plasma and PBMCs will be collected in four 10mL EDTA                                                                                                                                                                                                 |
| App - 1C                        |                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| AMC 097 (Version 9.0) 07FEB2022 | ii                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |  
AMC 097 (Version 9.0) 07FEB2022 ii  
NCI Version Date 07FEB2022  
CLIENT CONFIDENTIAL  
| #                                                                                                     | Section   | Description of Change                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
|-------------------------------------------------------------------------------------------------------|-----------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| tubes and cryopreserved at the time of ART resumption and at weeks 4,  8 and 24 after restarting ART. |           |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| 7.                                                                                                    | 3.1.2.3   | Inclusion criterion was added for HIV viral load requirements, which  include options for patients with higher viral loads if it can be determined  which antiretroviral regimen can suppress the virus, as this is more  consistent with clinical practice.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |
| 8.                                                                                                    | 4.2.1     | Dosing recommendations for BEAM and R-BEAM therapy have been  eliminated to permit the use of biosimilars for rituximab, as sites are  permitted to administer therapy per standard of care and their local  institutional guidelines. In addition, rituximab biosimilars inclusion  enables an alternative option in the event there is a supply shortage of  rituximab at their institution.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |
| 9.                                                                                                    | 4.7       | Criteria for withdrawing from the study was revised. "Progression of  Lymphoma" was removed as a criterion and participants will instead be  followed for correlative studies as outlined in Section 7.5.8.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| 10.                                                                                                   | 7.5.6     | The protocol has been updated to include optional bone marrow biopsy  collections at 3 months, 12 months, and at study discontinuation for  participants that do not have bone marrow involvement at baseline and  that consent to optional studies. In addition, clarifications were added to  distinguish requirements / purpose of required (restaging for those with  bone marrow involvement) vs optional bone marrow biopsies (for  research).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| 7.5.8.7 7.6.1.6  7.6.2.6                                                                              |           |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| App - 1B App - 1D                                                                                     |           |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| Appendix VII                                                                                          |           |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| 11.                                                                                                   | 7.5.8.2   | The University of California San Francisco (UCSF) is conducting the  SCOPE study which includes an optional GI biopsy at the same  timepoints as this study. The SCOPE study is an observational,  prospective study of HIV-1 infected volunteers designed to provide a  specimen bank of samples with carefully characterized clinical data. Specimens banked as part of the SCOPE study will be used to examine  questions involving virologic, immunologic, and host factors involved in  HIV-1 infection, progression, non-progression, response to treatment,  control of HIV-1 virus, and evolution of drug resistance. Participants that  agree to enroll into both the SCOPE study and AMC-097 at the same time (co-enroll) and agree to optional tissue collections will have their biopsy  results shared for both studies to reduce the number of biopsies performed  and facilitate enrollment to these optional studies. |
| AMC 097 (Version 9.0) 07FEB2022                                                                       | iii       |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |  
AMC 097 (Version 9.0) 07FEB2022 iii  
NCI Version Date 07FEB2022  
CLIENT CONFIDENTIAL  
| #                 | Section   | Description of Change                                                                                                                                                                                                              |
|-------------------|-----------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 12.               | 7.7       | Follow-up requirements have been revised to remove UC Davis as the  responsible party for contacting participants and/or their providers during  study required follow-up. Each site will contact the participants they  enrolled. |
| App - 1B          |           |                                                                                                                                                                                                                                    |
| 13.               | App - 1B  | Added optional blood sample collections every 12 months for years 3-15  for HIV viral load, and DNA and immunologic correlative studies to  monitor participant's HIV and vector parameters in the body.                           |
| App - 1D 4.6  7.7 |           |                                                                                                                                                                                                                                    |
summary of changes
iii. administrative and editorial changes

| #                               | Section             | Description of Change                                                                                                                                                                                                                                                                                                                                                        |
|---------------------------------|---------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 14.                             | Global              | The protocol version was changed from 8.0 to 9.0 and the date from  10SEP2020 to 07FEB2022.                                                                                                                                                                                                                                                                                  |
| 15.                             | Global              | Editorial and administrative changes were applied to spelling, grammar, punctuation, and table/figure numbering.                                                                                                                                                                                                                                                             |
| 16.                             | Global              | Replaced "subject" with "participant" throughout.                                                                                                                                                                                                                                                                                                                            |
| 17.                             | Protocol  Synopsis  | The definition of ANC and Platelet engraftment has been clarified to  include the underscore line with the greater than symbol. The ">"  (greater than) is changed to '>/=' (greater than or equal) to be consistent  with other sections of the protocol which state 'absolute neutrophil count  of at least 500 cells/mm3  and platelet count of at least 20,000 cells/mm3 |
| 1.3.2.5 1.3.2.6 2.3 9.1         |                     |                                                                                                                                                                                                                                                                                                                                                                              |
| 18.                             | 2.4.1               | Subsections for ART resumption criteria, ART monitoring, and postART monitoring have been added for clarity.                                                                                                                                                                                                                                                                                                                                                                              |
| 2.4.2 2.4.3                     |                     |                                                                                                                                                                                                                                                                                                                                                                              |
| 19.                             | 3.5                 | Corrected the EDC participant ID obtained after registration from nine  digit to eleven digits.                                                                                                                                                                                                                                                                              |
| Appendix  VII                   |                     |                                                                                                                                                                                                                                                                                                                                                                              |
| 20.                             | 5.1                 | Removed definition of CD4+T cells for brevity.                                                                                                                                                                                                                                                                                                                               |
| AMC 097 (Version 9.0) 07FEB2022 | iv                  |                                                                                                                                                                                                                                                                                                                                                                              |
| NCI Version Date 07FEB2022      | CLIENT CONFIDENTIAL |                                                                                                                                                                                                                                                                                                                                                                              |  
| #   | Section     | Description of Change                                                                                                                                                                                                                                                                                                                                                                                                            |
|-----|-------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 21. | 7.4.2       | Updated conditioning therapy to include R-BEAM as an option for  consistency with Section 4.2.1.                                                                                                                                                                                                                                                                                                                                 |
| 22. | Appendix IApp - 1D             | Sample collections for CCR5-Tropism were added for consistency with  the screening evaluations in Section 7.1. Collections for immunology  studies were updated to indicate specimens are to be collected in four  10mL EDTA tubes and one 5 mL redtop tube at 3, 6, and 12 months posttransplant and treatment discontinuation for consistency with Appendix  VII. In addition, footnotes were added for GI biopsy collections to reflect  that an specimen collections are optional and that GI biopsies may be  delayed until the participant's CD4 count has reached 300 cells/mm3 and  the biopsy repeated 2 months after the first collection for consistency with  Section 7.5.8.2 and Appendix I.                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| 23. | Appendix VI | AMC Data and Safety Monitoring Plan was updated to version 9.0. Key  revisions include revision to state that the IRB review plan is identified  in the protocol, and will continue to reference situations that apply for  both domestic and international trials, as the AMC is preparing to open  international trials where the requirements for US IRB review and  international IRB review will be specific to the region. |  
AMC PROTOCOL \#097:  
  
A Phase I Study of Stem Cell Gene Therapy for HIV Mediated by Lentivector Transduced, Pre-Selected CD34+ Cells A Trial of the AIDS Malignancy Consortium (AMC)  
| Sponsored by:                  | National Cancer Institute Office of HIV and AIDS Malignancy (OHAM)   |
|--------------------------------|----------------------------------------------------------------------|
| NCT Registration               | NCT02797470                                                          |
| Number: Investigational Agent: | Autologous CD34+ cells transduced with a preselective marker and triple combination of antiHIV genes (NSC 784725)                                                                      |
| IND #:                         | 16129 Sponsor-Investigator: Mehrdad Abedi, MD                        |
| Protocol Chair:                | Mehrdad Abedi, MD                                                    |
| Protocol Co-Chair:             | Joseph Anderson, PhD Ariela Noy, MD                                  |
| Version 9.0, 07FEB2022         |                                                                      |
| NCI Version Date 07FEB2022     |                                                                      |
summary of changes
amc protocol signature page

I, **____________________**, Principal Investigator at site ________, agree to conduct and follow this protocol: AMC Protocol \# 097 - A Phase I Study of Stem Cell Gene Therapy for HIV
Mediated by Lentivector Transduced, Pre-Selected CD34+ Cells (Version 9.0, 07FEB2022), as written according to AMC, NCI, International Conference on Harmonization guideline for Good Clinical Practice E6, the Declaration of Helsinki, and FDA guidelines. I understand that no deviations from the protocol eligibility criteria or waivers for protocol deviations will be permitted. _________________________________ ______________________ Signature Date (DDMMMYYYY)  
| TABLE OF CONTENTS                                                                                                                                    |                                                                                                                                                       |
|------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------|
| SUMMARY OF CHANGES .                                                                                                                                 |                                                                                                                                                       |
| I.                                                                                                                                                   | Recommendations from Approval with Recommendations Memorandum                                                                                         |
| for Protocol Version 8.0 (dated 10SEP2021) from R. Little, dated                                                                                     |                                                                                                                                                       |
| 24SEP2020:                                                                                                                                           |                                                                                                                                                       |
| II.                                                                                                                                                  | Scientific and Substantive Changes:                                                                                                                   |
| III.                                                                                                                                                 | Administrative and Editorial Changes:                                                                                                                 |
| AMC PROTOCOL SIGNATURE PAGE                                                                                                                          |                                                                                                                                                       |
| TABLE OF CONTENTS                                                                                                                                    |                                                                                                                                                       |
| LIST OF TABLES AND FIGURES .                                                                                                                         |                                                                                                                                                       |
| PROTOCOL ROSTER                                                                                                                                      |                                                                                                                                                       |
| PROTOCOL SYNOPSIS                                                                                                                                    |                                                                                                                                                       |
| PROTOCOL SCHEMA                                                                                                                                      |                                                                                                                                                       |
| LIST OF ABBREVIATIONS                                                                                                                                |                                                                                                                                                       |
| 1.0                                                                                                                                                  | OBJECTIVES .                                                                                                                                          |
| 1.1                                                                                                                                                  | Hypothesis                                                                                                                                            |
| 1.2                                                                                                                                                  | Primary Objective ....................................................................................................................18              |
| Secondary Objectives                                                                                                                                 |                                                                                                                                                       |
| 1.3                                                                                                                                                  |                                                                                                                                                       |
| 1.4                                                                                                                                                  | Exploratory Objectives ......................................................................................................19                       |
| 2.0                                                                                                                                                  | BACKGROUND .                                                                                                                                          |
| 2.1                                                                                                                                                  | Study Disease                                                                                                                                         |
| 2.2                                                                                                                                                  | Study Agent: Autologous CD34+ Cells Transduced with a Pre-Selective                                                                                   |
| Marker and a Triple Combination of Anti-HIV Genes                                                                                                    |                                                                                                                                                       |
| 2.3                                                                                                                                                  | Study Design and Rationale...................................................................................................23                       |
| ART Withdrawal / Analytic Treatment Interruption (ATI)                                                                                               |                                                                                                                                                       |
| 2.4                                                                                                                                                  |                                                                                                                                                       |
| 2.5                                                                                                                                                  | Correlative Studies                                                                                                                                   |
| 3.0                                                                                                                                                  | PARTICIPANT SELECTION .............................................................................................................................49 |
| 3.1                                                                                                                                                  | Eligibility Criteria .............................................................................................................49                  |
| Exclusion Criteria ...............................................................................................................................52 |                                                                                                                                                       |
| 3.2                                                                                                                                                  |                                                                                                                                                       |
| 3.3                                                                                                                                                  | Number of Participants to be Enrolled.....................................................................................53                          |
| 3.4                                                                                                                                                  | Recruitment Methods                                                                                                                                   |
| Participant Enrollment Procedures ..............................................................................................................55   |                                                                                                                                                       |
| 3.5                                                                                                                                                  |                                                                                                                                                       |
| 4.0                                                                                                                                                  | TREATMENT PLAN                                                                                                                                        |
| 4.1                                                                                                                                                  | HSC Mobilization and Peripheral Stem Cell Collection ............................................56                                                   |
| AMC-097 (Version 9.0) 07FEB2022                                                                                                                      | 3                                                                                                                                                     |
| NCI Version Date 07FEB2022                                                                                                                           | CLIENT CONFIDENTIAL                                                                                                                                   |  
| 4.2   | Agent Administration 57                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
|-------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 4.3   | Definition of Dose-Limiting Toxicity 59                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| 4.4   | General Concomitant Medication and Supportive Care Guidelines 60                                                                                                                                                                                                                                                                                                                                                                                                                     |
| 4.5   | Duration of Therapy 60                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| 4.6   | Duration of Follow Up 61                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
| 4.7   | Withdrawal of Participants 61                                                                                                                                                                                                                                                                                                                                                                                                                                                        |
| 5.0   | ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS 62 5.1 Comprehensive Adverse Events and Potential Risks Lists 62 5.2 Classification of AEs by Severity and Relationship to Study Drug  Administration 64 5.3 Expedited Adverse Event Reporting 64 5.4 Routine Adverse Event Reporting 68 5.5 Clinical Laboratory Results 68 5.6 Secondary Malignancy 68 5.7 Second Malignancy 69                                                                                                     |
| 6.0   | PHARMACEUTICAL INFORMATION 70 6.1 Autologous CD34+ Cells Transduced with a Pre-selective Marker and  Triple Combination of Anti-HIV Genes, IND Agent (NSC # 784725) 70                                                                                                                                                                                                                                                                                                               |
| 7.0   | CLINICAL AND LABORATORY EVALUATIONS 77 7.1 Screening Evaluations Required Within 8 Weeks Prior to Stem Cell  Collection (unless otherwise noted) 77 7.2 Screening Evaluations required within 1 week of admission for stem cell  transplant 78 7.3 Required on the day of admission for transplant prior to start of  chemotherapy 79 7.4 Evaluations During Treatment 79 7.5 Post-Treatment Follow-up Evaluations 79 7.6 Discontinuation Evaluations 82 7.7 Post-Study Follow-up 83 |
| 8.0   | MEASUREMENT OF EFFECT 84 8.1 Response Parameters 84 8.2 Frequency of Response Evaluation 84 8.3 Safety Measures 84                                                                                                                                                                                                                                                                                                                                                                   |
| 9.0   | STATISTICAL CONSIDERATIONS 85                                                                                                                                                                                                                                                                                                                                                                                                                                                        |  
| 9.1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Study Design/Endpoints 85                                                                                                                                                                                                                                                                                                                                                                 |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 9.2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Sample Size/Accrual Rate 86                                                                                                                                                                                                                                                                                                                                                               |
| 9.3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Early Stopping Rules 86                                                                                                                                                                                                                                                                                                                                                                   |
| 9.4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Cohort Escalation Design 86                                                                                                                                                                                                                                                                                                                                                               |
| 9.5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Analysis of Secondary Endpoints 86                                                                                                                                                                                                                                                                                                                                                        |
| 9.6                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | AMC Policy for Monitoring of Phase II Trials 87                                                                                                                                                                                                                                                                                                                                           |
| 10.0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | ROLE OF DATA MANAGEMENT 89 10.1 CRF Instructions 89 10.2 Data Quality 89 10.3 Data Monitoring 89                                                                                                                                                                                                                                                                                          |
| 11.0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | ETHICAL AND REGULATORY CONSIDERATIONS 90 11.1 IRB Approval and Informed Consent 90 11.2 Multi-Site Research 90 11.3 Data Management and Confidentiality 91 11.4 Changes to the Protocol 92 11.5 Vulnerable Populations 92 11.6 Non-English-Speaking Participants 92 11.7 Women and Minorities 92 11.8 Compensation for Research-Related Injury 93 11.9 Economic Burden to Participants 93 |
| 12.0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | REFERENCES 94                                                                                                                                                                                                                                                                                                                                                                             |
| APPENDIX I: SCHEDULE OF EVALUATIONS 97 App - 1A: Pre-Transplant Screening; Mobilization Period 97 App - 1B: Post-Transplant Follow Up 99 App - 1C: Analytic Treatment Interruption 101 App - 1D: Sample Collection guidance 103 APPENDIX II: PERFORMANCE STATUS SCALES 105 APPENDIX III: AIDS AND CANCER SPECIMEN RESOURCE (ACSR) SPECIMEN  PREPARATION AND SHIPPING INSTRUCTIONS 106 APPENDIX IV: ACSR INFORMED CONSENT 109 APPENDIX V: CONSENT FORM FOR GI BIOPSIES 114 APPENDIX VI: AMC DATA AND SAFETY MONITORING PLAN 120 APPENDIX VII: CORRELATIVE STUDY SPECIMEN PREPARATION AND SHIPPING |                                                                                                                                                                                                                                                                                                                                                                                           |  
| INSTRUCTIONS 125                                                                                                                                                                                                                                                                                                                     |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| APPENDIX VIII: INTERNATIONAL PROGNOSTIC INDEX (AGE-ADJUSTED) 137 APPENDIX IX: CCR5-TROPISM ASSAY 138 APPENDIX X: RISK LIST FOR BEAM 139 APPENDIX XI: ATI RISK MITIGATION COUSENLING FORMS: 141 APPENDIX XII: HIV TREATMENT INTERRUPTION PARTICIPANT DISCLOSURE  FORM: 143 APPENDIX XIII: PREP INFORMATION SHEET FOR PARTICIPANTS 144 |
summary of changes
list of tables and figures

Table 2-A: Study Cohorts ................................................................................................................. 24 Figure 2-B: Enrichment of 1TAX vector transduced cells: Human CD34+ HPC were transduced with the 1TAX vector, purified by CD25 immunomagnetic beads, and analyzed by flow cytometry for CD25 expression. Non‐ transduced cells (NT), flow through (ft) .................. 32 Figure 2-C: Down regulation of CCR5 expres**sion on 1TAX cells: Ghost‐R5/X4/R3 cells** transduced with the 1TAX vector were analyzed by flow cytometry for CCR5 cell surface expression. A representative histogram is displayed with NT cells (unshaded) and 1TAX vector transduced cells (shaded). Experiments were performed in at least triplicate. *** p<0.001 ...... 33 Figure 2-D: Phenotypic profile of 1TAX vector transduced peripheral blood T cells: Human CD34+ HSC were transduced with the 1TAX vector and transplanted into NRG mice. The peripheral blood of engrafted mice was analyzed by multi-parametric flow cytometry for T cell phenotypes .......................................................................................................................................... 34 Figure 2-**E: HIV‐1 challenge of 1TAX HSC derived macrophages: A) Nontransduced (NT) (♦**) and 1TAX vector transduced (■) HSC derived macrophages were challenged with a CCR5tropic BaL-1 strain of HIV-**1. On multiple days post‐infection, cell culture supernatants were**
sampled and assessed for HIV-1 using a p24 antigen ELISA kit. Experiments were performed in triplicate. * p<0.05, ** p<0.01, *** p<0.001 ................................................................................ 35 Figure 2-F: Maintenance of human CD4+ T cells in vivo in 1TAX cell engrafted NRG mice infected with HIV‐1: NRG mice engrafted with either untransduced **(NT) or 1TAX vector** transduced cells were infected intraperitoneally with a (A) CCR5‐tropic BaL‐1 or a (B) CXCR4‐tropic NL43 strain of HIV‐1. Various weeks post‐infection, mice were bled via tail vein and analyzed by flow cytometry for total human CD3+/CD4+ cell percent. Bar graphs display averages and standard deviations from seven mice per cohort for the BaL‐1 infections and four mice per cohort for the NL43 infections for CD3+/CD4+ cell levels pre-infection and 15 weeks post‐infection. * p<0.05 and ** p<0.002 ........................................................................................... 37 Figure 2-G: Reduction in HIV-1 plasma viremia in NRG mice engrafted with 1TAX vector transduced HSC: NRG mice engrafted with either untransduced (NT) or 1TAX vector transduced cells were infected intraperitoneally with a (A) CCR5‐tropic BaL‐1 or a (B)
CXCR4-tropic NL43 strain of HIV‐1. Various weeks post**-infection, mice were bled via tail vein** and analyzed by qPCR for plasma viremia using a primer/probe set specific for the HIV LTR. Log RNA copies/mL for NT (**♦) (solid grey lines), 1TAX vector transduced (■) (solid black** lines), and background levels from uninfected (UI) mice (▲) (dashed grey lines). Line graphs display average**s and standard deviations from seven mice per cohort for the BaL‐1 infections** and four mice per cohort for the NL43 infections. * p<0.05 and ** p<0.005. .............................. 38  
| and four mice per cohort for the NL43 infections. * p<0.05 and ** p<0.005 38 Table 4-A: Dose of Transduced Versus Untransduced Cells 57 Table 4-B: Criteria for dose escalation 59 Table 7A: Follow-Up Schedule 79 Table 11-A: Accrual Targets 93   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
The schedule during the ATI period will be defined by clinical course and the characteristics of any virus rebound. ART will be initiated for clinical events, sustained drop in CD4+T cell counts or sustained increased in viremia as described in Section 2.4.1. Prior to these events, participants will be followed. If collection timepoints for CD4/CD8 and HIV VL overlap with collections as part of the Follow-up Evaluations, then they do not need to be repeated. Clinical laboratory testing, assessments, and ATI counseling may be performed remotely to minimize in-person visits during the ATI. ..................................................................................................... 101
summary of changes
protocol roster

AMC Protocol \# 097 A Phase I Study of Stem Cell Gene Therapy for HIV Mediated by Lentivector Transduced, Pre-Selected CD34+ Cells
summary of changes
protocol chair

Mehrdad Abedi, MD University of California Davis Comprehensive Cancer Center 4501 X Street Sacramento, CA 95817 Tel: (916) 734-3772 Fax: (916) 734-7946 Email: mabedi@ucdavis.edu
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protocol co-chair

Joseph Anderson, PhD University of California Davis 2921 Stockton Blvd. Room 1300 Sacramento, CA 95817 Tel: 916-703-9300 Fax: 916-703-9310 Email: jsanderson@ucdavis.edu
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protocol co-chair ariela noy md

Memorial Sloan-Kettering Cancer Center Lymphoma Service 1275 York Avenue New York, NY 10065 Tel: (212) 639-7423 Fax: (646) 422-2284 Email: noya@mskcc.org
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amc biorepository director

Sylvia Silver, DA George Washington University Medical Center AMC Biorepository Ross Hall, Room 118 2300 I St, NW Washington, DC 20037 Tel: (202) 994-2945 Fax: (202) 994-5056 Email: ssilver@gwu.edu AMC-097 (Version 9.0) 07FEB2022 8 NCI Version Date *07FEB2022* CLIENT CONFIDENTIAL
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protocol statistician

Jeannette Lee, PhD University of Arkansas for Medical Sciences 4301 West Markham Street Tel: 501-526-6712 Fax: 501-526-6729 Email: jylee@uams.edu
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data management/operations

AMC Operations and Data Management Center The Emmes Company, LLC 401 N. Washington Street, Suite 700 Rockville, MD 20850 Tel: (301) 251-1161 Fax: (240) 238-2842 Email: amcpm@emmes.com
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amc hematologic malignancy working group

Richard Ambinder, MD, PhD Oncology Center Bunting-Blaustein Cancer Research Building 1650 Orleans Street, Room 389 Baltimore, MD 21231Tel: (410) 955-8839 Fax: (410) 955-0960 Email: ambinri@jhmi.edu
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protocol synopsis

Title: A Phase I Study of Stem Cell Gene Therapy for HIV Mediated by Lentivector Transduced, Pre-Selected CD34+ Cells Phase of Study: Phase I Methodology Open label Participating Institutions:
This protocol will be open initially to UC Davis, UC San Diego, and UC San Francisco Medical Centers. Upon completion of the first cohorts additional AMC sites will be added dependent on the recruitment rate. A decision on any additional participating trial sites will be made after consultation with the AMC Hematologic Malignancy Working Group.  
Memorial Sloan Kettering Cancer Center was added as a site in AUG
2019.  
Accrual Target: This study will enroll a minimum of 2 participants and a maximum of 18 participants.  
| Population:   | Male or female participants greater than 18 years of age, with HIV-1  related lymphomas that are refractory/resistant to first line of therapy or  relapse after achieving a remission and are eligible for an autologous  HSCT as a part of their routine therapy are eligible for this study.  Participants must be on a multi-drug anti-HIV regimen (excluding  zidovudine [AZT, ZDV, Retrovir®, or agents containing zidovudine  (e.g., Combivir® and Trizivir®)] and efavirenz [EFV, Sustiva®, or agents containing efazirenz (e.g., Atripla®)]) and have an HIV-1 viral load < 50  copies/mL by RT-PCR at the time of study enrollment.   |
|---------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Regimen: For the first 2 cohorts, both transduced (autologous CD34+ cells transduced with a pre-selective marker and a triple combination of antiHIV genes) and un-manipulated product will be infused within 4 to 24 hours of each other. After DSMB approval, only transduced product will be administered to the rest of the participants. In case of nonengraftment, the back-up product stored at the respective site will be infused. The number of cells infused to each participant depends on the cohort in which the participant is enrolled. The number of cells in the transduced products is based on CD34 counts after transduction of the stem cells and before freezing.  
| Minimum Number of  Transduced/Untransduced Cells  Acceptable. CD34  cells/kg                                          |                                                                      |                  |                              |
|------------------------------------------|----------------------------------------------------------------------|------------------|------------------------------|
| Ratio of                                 |                                                                      |                  |                              |
| Transduced vs  Un-transduced  Stem Cells | Range for Number of  Transduced  Cells/Unmanipulated.  CD34 cells/kg |                  |                              |
| Study  Cohorts 1                         | 1:1                                                                  | 1 X 106 :1 X 106 | 1 to 5 X 106 : 1 to 5 X  106 |
| 2                                        | 5:1                                                                  | 2 X 106 :1 X 106 | 2 to 5 X 106 :1 X 106        |  
AMC-097 (Version 9.0) 07FEB2022 9  
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| 3                  | 1:0                                                                                                                                                                                                       | 2 X 106 : 0   | 2 to 10 X 106 : 0   |
|--------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|---------------------|
| 3+                 | 1:0                                                                                                                                                                                                       | 3.0 X 106 : 0 | 3.0 to 10 X 106 : 0 |
| Duration:          | The initial follow-up period for this clinical trial will be two years;  however, all participants will be followed for 15 years for safety, as  mandated by FDA for all gene therapy study participants. |               |                     |
| Primary Objective: | To evaluate the clinical safety of our approach using our development  candidate (i.e., anti-HIV gene modified hematopoietic stem cells). See  Section 1.2 for endpoint definitions.                      |               |                     |
| Secondary          | To evaluate immune reconstitution with HIV-1 resistant immune cells,                                                                                                                                      |               |                     |
| Objectives:        | viral load and HIV-1 status, integration sites of vector sequences in  circulating cells, presence of any in vivo clonal expansion of transduced  cells, and any immune reaction to transduced cells.     |               |                     |
summary of changes
major secondary objectives

- To determine the efficacy of our approach using our development candidate.  
- To determine the presence, quantity, and duration of gene modified HIV-1 resistant peripheral blood cells and gut mucosal immune cells.  
- To study the integration sites of vector sequences in circulating cells.
summary of changes
minor secondary objectives

- To study progression-free survival.  
- To study overall survival.  
- To study complete response rate and duration.  
- To study partial response rate and duration.  
- To study time to neutrophil engraftment (first measurement of 3 consecutive laboratory values obtained on different days) of ANC >
/= 500 cells/mm3.  
- To study time to platelet engraftment (first measurement of 3 consecutive laboratory values obtained on different days) of platelets
> /= 20,000 cells/mm3 without platelet transfusions 7 days prior).
- To study hematologic function at Day 100 (ANC > 1500, Hb >
10g/dl without transfusion and platelets > 100,000)
- To study CD4 recovery at the conclusion of the trial.  
- To study safety in terms of toxicities, infections, transfusions, and infusion-related reactions.  
- To study HIV-1 viral load over time.
- To study persistence of vector-transduced cells over time.  
| To explore the role of anti-retroviral therapy (ART) interruption on  expansion of HIV-1 resistant HIV-1 target cells, including immune cells,  both in the peripheral blood and the gut mucosa.  Three participants will be enrolled in each cohort with potential  expansion of the cohort to 6 with the maximum of 18 participants.  The primary endpoint of the study is safety.  Safety is defined as timely engraftment (the collective establishment of  a persistent absolute neutrophil count of at least 500 cells/mm3 and  platelet count of at least 20,000 cells/mm3 for 3 consecutive  measurements of laboratory values obtained on different days) within one month post-transplant, in the absence of any study candidatespecific grade 3 and 4 non-hematopoietic organ toxicity, excluding   |
|---|  
  
| LIST OF ABBREVIATIONS           |    |
|---------------------------------|----|
| ACSR .                          |    |
| A A VANTAG                      |    |
| AE .                            |    |
| AIDS .                          |    |
| AMC                             |    |
| AML.                            |    |
| ANC                             |    |
| ART.                            |    |
| ATI.                            |    |
| AZT                             |    |
| BCNU .                          |    |
| BEAM .                          |    |
| R-BEAM .                        |    |
| BID.                            |    |
| BSA                             |    |
| BMT                             |    |
| CC                              |    |
| CR5.                            |    |
| CDUS .                          |    |
| CEF                             |    |
| CFR                             |    |
| CFSE .                          |    |
| CFU.                            |    |
| CI                              |    |
| cm                              |    |
| CMP                             |    |
| CMV                             |    |
| CNS                             |    |
| COA                             |    |
| CR .                            |    |
| CRF                             |    |
| CT .                            |    |
| CTA.                            |    |
| CTCAE                           |    |
| CTEP .                          |    |
| CTEP-AERS                       |    |
| CTL                             |    |
| CTMS                            |    |
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| CR4.                            |                     |
|---------------------------------|---------------------|
| CXR                             |                     |
| DARF .                          |                     |
| DC .                            |                     |
| DHS.                            |                     |
| DLCO                            |                     |
| DLT.                            |                     |
| DMSO .                          |                     |
| DNA                             |                     |
| DSMB .                          |                     |
| EBMT .                          |                     |
| EV                              |                     |
| ECOG                            |                     |
| EDTA .                          |                     |
| EFV                             |                     |
| EGFP .                          |                     |
| ELISA                           |                     |
| EU/mL.                          |                     |
| FACS                            |                     |
| FACT .                          |                     |
| FBS                             |                     |
| FDA.                            |                     |
| FDG-PET                         |                     |
| FEV-1                           |                     |
| ft .                            |                     |
| FVC.                            |                     |
| g/dl .                          |                     |
| GALT .                          |                     |
| GAPDH                           |                     |
| GCP                             |                     |
| GI .                            |                     |
| GMP                             |                     |
| HAV                             |                     |
| Hb                              |                     |
| HIV                             |                     |
| HCT.                            |                     |
| HIPAA                           |                     |
| HLA                             |                     |
| HPC.                            |                     |
| HSC.                            |                     |
| AMC-097 (Version 9.0) 07FEB2022 | l 4                 |
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| HSCT .                          |                     |
|---------------------------------|---------------------|
| HV                              |                     |
| HTLV .                          |                     |
| ICH.                            |                     |
| IDB.                            |                     |
| IFN                             |                     |
| IHC.                            |                     |
| IL .                            |                     |
| IND                             |                     |
| INR.                            |                     |
| IPI                             |                     |
| IRB                             |                     |
| kg                              |                     |
| L LAL                           |                     |
| LN .                            |                     |
| LAM-PCR.                        |                     |
| LDH                             |                     |
| LTR                             |                     |
| LVEF                            |                     |
| MCP                             |                     |
| mm                              |                     |
| MP .                            |                     |
| µµ                              |                     |
| MDS                             |                     |
| mL                              |                     |
| m 3                             |                     |
| MOI                             |                     |
| MOP                             |                     |
| mRNA .                          |                     |
| MUGA .                          |                     |
| NCI.                            |                     |
| NCT.                            |                     |
| ng                              |                     |
| NIH.                            |                     |
| NHL.                            |                     |
| NK cells .                      |                     |
| NRG mouse.                      |                     |
| NC                              |                     |
| NT .                            |                     |
| OMC.                            |                     |
| AMC-097 (Version 9.0) 07FEB2022 | 15                  |
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| OHAM .                          |                     |
|---------------------------------|---------------------|
| O                               |                     |
| O                               |                     |
| PB                              |                     |
| PBMC .                          |                     |
| PCP                             |                     |
| PCR                             |                     |
| PDL                             |                     |
| PET                             |                     |
| PFS                             |                     |
| PFT.                            |                     |
| PHI                             |                     |
| PI .                            |                     |
| PIO                             |                     |
| PT .                            |                     |
| PTT                             |                     |
| QC                              |                     |
| QA                              |                     |
| qPCR                            |                     |
| RAC                             |                     |
| RCL.                            |                     |
| RNA                             |                     |
| RR                              |                     |
| RT-PCR                          |                     |
| SE SE                           |                     |
| SOC.                            |                     |
| SD .                            |                     |
| SEB                             |                     |
| SGOT .                          |                     |
| SGPT                            |                     |
| SMART.                          |                     |
| SOP                             |                     |
| shRNA.                          |                     |
| STI.                            |                     |
| TAR.                            |                     |
| TAX (1TAX) .                    |                     |
| Tat                             |                     |
| TNFα .                          |                     |
| TRIM5α .                        |                     |
| TR .                            |                     |
| AMC-097 (Version 9.0) 07FEB2022 | 16                  |
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| T Treg .   |
|------------|
| VDRL       |
| VV .       |
| ULN        |
| USP        |
| ZDV        |
summary of changes
1.0 objectives 1.1 hypothesis

Our hypothesis states that anti-HIV gene expressing stem cells can safely reconstitute the hematopoietic system of the recipient and can produce a pool of mature myeloid and lymphoid cells that harbor anti-HIV genes capable of resisting HIV-1. As a secondary objective, we will interrogate two physical compartments of immune system, (i.e., peripheral blood cells and gut mucosa) for the presence, quantity, and duration of gene modified HIV-1-resistant immune cells. In addition, we will determine the integration sites of vector sequences in circulating cells and monitor for any clonal expansion of hematopoietic cells. We also hypothesize that transduced HIV-resistant immune cells will have a selective survival advantage as compared to non-transduced cells in the face of a viral load, as demonstrated by our preclinical *in vivo* experiments, and suggested by previous clinical trials using comparable stem cell gene therapy approaches. As an exploratory endpoint, we therefore want to test the efficacy of our stem cell gene therapy for HIV-1 in the context of structured HIV-1 treatment interruptions.
summary of changes
1.2 primary objective

The primary endpoint of the study is safety, defined as timely engraftment (the collective establishment of a persistent absolute neutrophil count of at least 500 cells/mm3 and platelet count of 20,000 cells/mm3 without transfusion for 3 consecutive measurements of laboratory values obtained on different days) by one month post-transplant, in the absence of any grade 3 and 4 non-hematopoietic organ toxicity that can be attributed (possibly, probably, or definitely) to lentiviral transduced stem cell transplant, excluding alopecia, or any clonal expansion and excluding expected toxicities that are associated with the pretransplant conditioning regimen (see Appendix X for full list).
summary of changes
1.3 secondary objectives

1.3.1 Major secondary objectives  
1.3.1.1 To determine efficacy of the candidate product, defined as establishment of > 5% mononuclear blood cells expressing anti-HIV genes in the peripheral blood at 3 months post-transplant.  
1.3.1.2. To determine the presence, quantity, and duration of gene modified HIV1 resistant peripheral blood cells and gut mucosal immune cells.  
1.3.1.3 To study the integration sites of vector sequences in circulating cells.
1.3.2 Minor secondary objectives 1.3.2.1 To study progression-free survival.  
1.3.2.2 To study overall survival. 1.3.2.3 To study complete response rate and duration. 1.3.2.4 To study partial response rate and duration. 1.3.2.5 To study time to neutrophil engraftment (first measurement of 3 consecutive laboratory values on different days) of ANC >/= 500 cells/mm3).  
1.3.2.6 To study time to platelet engraftment (first measurement of 3 consecutive measurements laboratory values obtained on different days) of platelets >
/=20,000 cells/mm3 without platelet transfusions 7 days prior).
1.3.2.7 To study hematologic function at Day 100 (ANC > 1500, Hb > 10g/dl without transfusion and platelets > 100,000)
1.3.2.8 To study CD4 recovery at the conclusion of the trial. 1.3.2.9 To study safety in terms of toxicities, infections, transfusions, and infusion-related reactions.  
1.3.2.10 To study HIV-1 viral load over time. 1.3.2.11 To study persistence of vector-transduced cells over time.
summary of changes
1.4 exploratory objectives

1.4.1 To evaluate the presence and the magnitude of expansion of HIV-1 resistant immune cells in the peripheral blood and gut mucosa of transplanted participants, subsequent to withholding ART.
summary of changes
2.0 background 2.1 study disease

HIV-1/AIDS continues to be a major public health problem worldwide. Based on recent estimations, there are approximately 33.3 million people living with HIV-1/AIDS with 2.6 million new HIV-1 infections per year, and 1.8 million annual deaths due to AIDS. It is estimated that more than one million people are living with HIV-1 in the USA and that more than half a million have died after developing AIDS. Despite the advances in antiretroviral therapy (ART), eradication of HIV-1 from infected patients is still an elusive goal. Lifelong therapy is associated with potential long-term toxicity, adherence problems, and development of drug resistance (1-6). Interruption of ART therapy at any stage will result in a rebound in HIV-1 replication and rapid deterioration of a patient's immune system. Even new drug classes such as integrase inhibitors and CCR5 antagonists, despite their promise, have failed to eradicate HIV-1 (5-6, 9-10). Patients who are successfully treated with ART often do not achieve full recovery of immune responses, may develop HIV-1-related cancers, and exhibit increased levels of immune activation (7-8). Such a chronic inflammatory status can result in immune exhaustion (and subsequent infections) and may precipitate multiple clinical complications such as cardiovascular and cerebrovascular disease, diabetes, chronic kidney disease, osteoporosis, and cancer, and collectively, accelerated aging. While the cause of this chronic inflammatory status is not completely clear, evidence suggests the role of innate immunity, ART interruption, noncompliance, effect of HIV-1 on endothelial cells, interruptions in the coagulation system, incomplete immune recovery, microbial translocation, co-infections, residual viremia, etc. 2.1.1 Gene therapy for HIV-1 patients with lymphoma Therefore, the idea of a cell-based gene therapy approach to cure HIV-1 would be especially compelling (11). Gene therapy has the potential to not only control viral replication and prevent CD4+ T cell depletion, but also provide a pool of immune cells with sustained intrinsic protection. HIV-1 gene therapy is not a new idea, and currently, gene therapy clinical trials are ongoing (12-14). However, there are major differences in the types of genes utilized as well as the cells targeted, which may be either peripheral blood T cells or hematopoietic stem cells (HSC). Gene therapy targets can be classified according to the same concept as applied to other anti-HIV-1 therapeutic strategies: Class I therapeutics inhibit HIV-1 entry and replication; Class II therapeutics inhibit viral regulatory and structural protein expression; and Class III therapeutics inhibit viral assembly and release. Most of the current anti-HIV gene therapy approaches (including our proposed protocol) focus on Class II and especially Class I gene targets that have been shown to perform as best available antivirals by far. Recently, a similar comparison has resulted in a similar conclusion in *in vivo* studies (15). While T cells are easy to obtain, their long-term engraftment is limited despite current lympho-depletion approaches. In addition, questions have recently been raised on the expansion of T cells in response to a high viral load (16-17). Furthermore, T cell gene therapy, despite its relative ease, cannot alleviate the main concerns about HIV-1 therapy such as the presence of low-level viral replication caused by dormant HIV-1 reservoirs. Therefore, the benefits of current T cell gene therapy protocols with the need for multiple, life long, continued cell infusions will not exceed the advantages of current ART therapy. Targeting human stem cells, as described here, on the other hand, can address many of these concerns. Such a stem cell therapy would require only a single treatment that would replace, in the long run; the reservoirs of active and latent HIV-1 with anti-HIV gene modified cells resistant to infection, thus alleviating the need for long-term ART and would ultimately cure these patients. In addition, unlike challenges in *in vivo* expansions of gene therapy modified T cells (as described above), anti-HIV gene modified stem cell derived HIV-1 target cells have shown that they do respond to an HIV-1 viral load with significant expansion of T cells (15,18). The best evidence supporting the feasibility of this comes from a case report of an HIV-1 patient transplanted with stem cells from a host homozygous for the CCR5 delta-32 mutation. The transplant resulted in reduction of HIV-1 viral load over time and the replacement of microglial and Kupffer cells, common reservoirs of latent HIV-1, by donor-derived HIV-1 resistant cells (18). This strongly suggests that the patient is now cured of HIV-1. Previous trials of HIV-1 gene therapy into HSCs have been plagued by low transduction efficiencies and loss/silencing of the anti-HIV genes over time (14, 18). Furthermore, single anti-HIV genes harbor the risk of developing resistant HIV-1 mutants (12). Finally, in some of these trials, the use of retroviral vectors (versus lentiviral vectors in our proposal) increases the risk of oncogenesis (13). Lessons learned from two recent computational models of HIV-1 replication and development of resistance to gene therapy regimens support our notion that the combinatorial anti-HIV gene therapy protocols and higher numbers of anti-HIV gene modified immune cells are critical factors in the success of gene therapy protocols for HIV-1 (19-20). Furthermore, they showed that when 1-2 anti-HIV targets are used, the success will be significantly dependent on the potency of the approach, while multiple combinatorial gene therapy approaches can eliminate such a requirement.
summary of changes
2.1.2 bone marrow transplant for

The prognosis for patients with refractory and relapsed non-Hodgkin's lymphoma (NHL) is poor with overall survival rates of less than 20% for patients treated with non-transplant salvage therapies. Based on these randomized trials, high-dose therapy with hematopoietic stem cell transplantation (HSCT) has been established as the standard of care for patients with relapsed but chemotherapy-sensitive NHL (21-23). This initial transplant experience was marked by what appeared to be an increased risk of opportunistic infection (OI). Since the availability of ART, however, the transplant experience has evolved considerably, and several groups have now demonstrated the feasibility of such an approach. In a trial published by City of Hope, patients with induction-failure NHL, chemotherapy-sensitive disease, and International Prognostic Index (IPI) high-risk disease were eligible for treatment. Twenty participants were enrolled in the trial. All of the participants successfully achieved adequate autologous stem cell collections. Participants were treated with either high-dose chemotherapy or intensive chemo-radiotherapy. At a median follow-up of 31.8 months, 17 of 20 participants were alive and free of evidence of disease. Treatment-related complications were comparable to those seen in the non-HIV-1 infected patient population (24).  
In a similar trial by the AIDS Malignancy Consortium (AMC), 27 participants with either HIV-1-related NHL or Hodgkin's lymphoma were enrolled and 20 of them subsequently underwent autologous hematopoietic cell transplantation (HCT) with a dose-reduced busulfan and cyclophosphamide regimen. At a median follow-up time of 23 weeks, 10 participants were alive and free of disease (25). In a recent trial by the AMC, 43 participants with HIV-1 CCSR aggressive NHL or Hodgkin's lymphoma were enrolled and 40 underwent autologous HCT using BEAM regimen (35). In this trial, 39 participants were alive 100 days post-HCT and 36 of the 39 of these participants were free of disease; and 24 of 32 participants achieved recovery of hematological function by 24 months post-HCT. Other groups have also demonstrated the feasibility of performing autologous HCT in this patient population (26-28). More recently, the European Bone Marrow Transplant (EBMT) Lymphoma Working Party, in a much bigger trial, published treatment outcomes for 68 participants with HIV-1-related lymphoma (including 18 participants with Hodgkin lymphoma) that underwent autologous HCT at 20 different institutions. Sixty-five participants were treated with the carmustine, etoposide, cytarabine, and melphalan (BEAM) regimen and the remainder were treated with radiation therapy-based regimens. At a median follow-up of 32 months, the 3-year estimated progressionfree and overall survivals for the group were 56% and 61% respectively, with low overall cumulative incidence of non-relapse mortality of 4.4% and 7.5% at three and 12 months respectively. Relapse or progression was identified in 20 participants after a median time of 4.1 months from autologous HCT with the predicted cumulative incidence of relapse of 30% at 24 months (29). A subsequent EBMT study for patients with Hodgkin's disease and non-Hodgkin's lymphoma used a comparison matched control design stratified by HIV-1 status. The results of this comparative study revealed that HIV-1 patients undergoing autologous HCT for lymphoma showed similar relapse rates, progression free survival, and overall survival (30). Based on these data, the practice of autologous HSCT is routinely used in many centers in the country as part of the treatment of relapsed/refractory (but still chemo-sensitive) NHLs.
summary of changes
2.2 study agent autologous cd34+

Our proposed novel anti-HIV therapeutic candidate combines multiple anti-HIV genes into a single lentiviral vector that is designed to block HIV-1 infection at different stages of the HIV-1 life cycle (entry, uncoating, and viral transcription). This vector also contains a preselective molecule, which marks transduced cells and allows for the purification/enrichment of the transduced population away from untransduced cells. This approach not only provides strong pre-integration inhibition of HIV-1 infection, but also decreases the generation of viral escape mutants. The major anti-HIV components of our vector are a CCR5 shRNA, a chimeric TRIM5α, and a TAR decoy. Due to the natural HIV1-resistant phenotype conferred by the delta-32 bp CCR5 mutation found in a small percent of humans, CCR5 knockdown utilizing shRNAs is an excellent approach for HIV-1 gene therapy. However, utilizing only a single CCR5 shRNA will not afford protection against CXCR4 or other tropisms of HIV-1. Therefore, we have incorporated two other highly potent anti-HIV genes into our lentiviral vector, which protect against all tropisms of HIV-1. The second molecule, a human/rhesus macaque chimeric TRIM5α molecule, acts at the post-entry/pre-integration stage of the HIV-1 life cycle. Old world monkey TRIM5α isoforms, which include the rhesus macaque isoform, are strong inhibitors of HIV-1 infection, however, human TRIM5α does not inhibit HIV-1. Therefore, we have utilized a chimeric isoform of human TRIM5α where we have incorporated the critical amino acids of the rhesus macaque isoform necessary to inhibit HIV-1 infection. This chimeric human TRIM5α now only contains 13 amino acids from the rhesus macaque isoform; however, it is now able to potently inhibit HIV-1 infection. The third molecule, a TAR decoy, acts at the pre-transcription stage of the HIV-1 life cycle and scavenges the HIV-1 Tat protein away from its normal function in HIV-1 proviral transcription. This removal of Tat from the transcriptional complex disrupts HIV-1 proviral transcription and does not allow new viral mRNA, and therefore, new virus particles to form. The pre-selective molecule, a truncated and mutated form of human CD25, is used to mark the cell surface of transduced cells, so they can be purified away from untransduced cells with CD25 immunomagnetic separation. This modified human CD25 molecule has been fully truncated of its intracellular amino acids to avoid any possible intracellular signaling. Two-point mutations have also been made to inhibit any IL2 binding, and also to inhibit the incorporation of this molecule into the mature IL2 receptor complex. The three anti-HIV molecules expressed together in a single lentiviral vector have demonstrated complete protection from productive HIV-1 infection and prevented integration of multiple strains of HIV-1-1 (including macrophage and T cell tropic HIV-1 strains) in both transduced cultured cells and transduced CD34+ HSCs derived into macrophages *in vitro* (17). *In vivo* safety and efficacy studies in a humanized mouse model for HIV-1 showed excellent results including maintenance of normal total human CD4+ cell levels with a pre-integration block of HIV-1 infection (18). Our preclinical *in-vitro* and in-vivo safety and efficacy data are mature enough to start the regulatory approval process for a stem cell gene therapy clinical trial. The successful development of a multi-gene preselective anti-HIV vector, excellent safety and efficacy results in both *in vitro* and *in vivo* testing of the vector transduced cells even for resistant strains of HIV-1, production of high titer GMP-grade lentiviral vector, and superior transduction efficiencies into HSCs are highlights of our proposed therapeutic candidate, which make it unique and more promising than other currently available gene therapy for HIV-1 approaches. Extensive preclinical data supporting this study is included in our IND document and the investigator's brochure.
summary of changes
2.3 study design and rationale

This is an open-label, single-arm, multi institution, Phase I study of maximum of 18 participants. The study is designed to fulfill the mission of a Phase I study as "a trial to study the safety, and determine the dose limiting toxicity (DLT) to a new treatment." All participants will be treated with anti-HIV gene transduced CD34+ cells.  
| Table 2-A: Study Cohorts Study Cohorts Ratio of Transduced versus Untransduced Stem  Cells 1 1:1 2 5:1 3 1:0 1:0 but with a higher transduced cell count only in  3+* case of DLT due to insufficient engraftment in  cohort 3  *. See section 2.3.1   |
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Three participants will be treated at each cohort level, with the option of 3-6 additional participants in the highest dose level cohort. Participants will be recruited to the groups in order that they were screened. Participants will be monitored on a weekly basis for the first 4 weeks and then every other week thereafter. A cohort will be expanded from 3 to 6 participants if at least one participant in the cohort experiences engraftment failure or grade 3 or 4 non-hematopoietic toxicity, excluding alopecia. The enrollment of the participants within the cohorts will be staggered so each participant needs to show at least early signs of engraftment for neutrophils (ANC) >/= 500 cells/mm3 and platelets >/= 20,000 cells/mm3 before the next participant is enrolled. The enrollment of participants between the cohorts will be staggered, so every participant in the same cohort needs to have at least 30 days of follow up from the time of stem cell transplant for the AMC Data and Safety Monitoring Board (DSMB) to review the side effects and before we can proceed to the next cohort. Achievement of high levels of engraftment with anti-HIV genes will be needed to secure a reasonable anti-HIV response. Therefore, in the third cohort, the participants will receive only the transduced product. The un-manipulated portion of product will remain frozen as a backup. In this cohort, the backup product will only be given to the participant if delayed engraftment occurs, especially in the setting of ongoing complications such as bleeding or infection. For safety reasons and based on FDA request, participants enrolled in the first cohort of the study will receive both transduced and un-transduced product at equal number. This is to prevent a prolonged cytopenia in case the transduced cells fail to engraft and recover the peripheral blood in a timely manner. In the second cohort, once the safety of the product is established, we will increase the ratio of the transduced cells to untransduced cells, with the goal to give 5 times (or higher) more transduced cells. Participants with relapsed/refractory HIV-1 related lymphoma in need of autologous bone marrow transplantation will be enrolled. Upon documentation of disease response, their peripheral stem cells will be collected using standard mobilization approaches. Because of the unpredictability of the number of cells collected for each patient and the significant variability of the number of cells obtained after transduction and selection process, a set of criteria has been created to explain the minimum and the range of cells infused to each patient (Table 4-A).  
- A target cell number of 10 x 106 CD34+ cells/kg will be collected, with a minimum of 5 x 106 CD34+ cells/kg only if we cannot collect enough cells after multiple attempts.  
- A minimum of 1.5 x 106 of unmanipulated CD34+ HSC/kg will be collected upfront and will be frozen in a minimum of 3 cryo bags and stored at each institution's local stem / progenitor cell facility per site SOPs. Even though the minimum amount of unmanipulated cells infused in the first two cohorts are 1.0 x 106 CD34+ cells/kg, additional 0.5 x 106 CD34+ cells/kg of recipient weight will be collected to ensure a safe transplant in the unlikely situation that the transduced product is not usable.  
- The minimum total number of CD34+ cells (manipulated + unmanipulated) infused to the patient is 2 x 106 CD34+ cells/kg of recipient weight independent of the cohort to assure the safety of transplant.  
- In cohorts 1 and 2, a minimum of 1 x 106 of unmanipulated CD34+ HSC/kg will be infused, and the rest of the cells will be stored as back-up. Additional CD34 cells from the transduced product given based on each cohort is expected. In cohort 3, all of the untransduced cells will be stored and used only in the case of delayed engraftment.  
- Additional cells can be collected and stored after the target number for CD34 (10 x 106 CD34+ cells/kg) is achieved based on the discretion of participant's transplant physician in consultation with the study principal investigator. These cells can be given to the patient, but the ratio of total manipulated to unmanipulated cells needs to follow the guidelines in Table 4-A.  
- In case of any failure in the transduction process, all of the unmanipulated cells will be infused to the patient.
Apheresis product, from the first day of collection, will be split into two parts: One part
(1.5 x 106 of CD34+ HSC/kg) will be cryopreserved (unmanipulated), and the second part, if additional cells are available, will be sent immediately to the GMP facility for gene transduction. We expect that we will need several days of collection for each participant.  
Once 1.5 x 106 CD34+ cells/kg is collected, then no more products will be divided for back up, and for the rest of the collections, the entire product from subsequent collections will be used for gene transduction. After transduction, this product will be frozen. Release tests will be performed on aliquots (see Section 6.1.11). 2.3.1 Three+ (3+) expansion cohort (protocol version 8)
The ultimate goal of the study is to replace the full hematopoietic system of the recipient with a gene modified anti-HIV hematopoietic system. The design of this clinical trial is different from other dose escalating clinical trials as we are not testing an increasing dose of our product and therefore routine phase I, 3+3 design is not applicable in this trial. The purified manipulated cells may not have the same robust engrafting capabilities as unmanipulated HSCs and, therefore, if in the third cohort 2 patients develop DLT due to insufficient engraftment, an additional 3 patients will be treated with a higher dose of gene-modified CD34 cells (3.0 x106 CD34 cells/kg of recipient body weight) to assess whether a higher dose of cells will improve engraftment. Any additional engraftment issue on this expanded cohort deemed to be related to product will result in stopping of the trial.
summary of changes
2.4 art withdrawal / analytic

The presence of sizable untransduced CD34 cells in the infused product will result in a population of hematopoietic cells in the participant after transplant that will remain sensitive to HIV-1 and still can drive a significant viral load with its associated complications. In the City of Hope clinical trial, significant expansion of HIV-1 resistant hematopoietic clone was observed when the participant was off his ART. Based on the above rationale and supporting observation, we are proposing to withdraw ART after 6 months post-transplant and when the CD4 counts reaches ≥ 300 cells/mm3.  
At six months post-transplant, or later, ART will be voluntarily withheld for participants with a CD4 count of 300 or higher with no detectable viral load (by single copy PCR) until one or more of the criterion in Section 2.4.1 prompting resumption of treatment. We do understand that the time to achieve a T-cell count of 300 may take longer than six months for HIV-1 infected participants post-transplant. Therefore, for participants in which the CD4 T-cell count has not risen to ≥ 300 cells/mm3 at the time of the planned ART interruption, ART will continue until the T-cell count has risen to ≥ 300 cells/mm3.  
In addition, participants will be tested for the virus that causes COVID-19 prior to ART interruption and if positive by PCR, will remain on ART until they recover, and the study team approves them to proceed with ATI. **Participants on ATI will be offered** COVID testing if symptoms **associated with COVID-19 develop** and will restart ART
if they test positive by PCR. The duration for ART withdrawal has been selected as it is an appropriate duration for assessment of any selective survival advantage of anti-HIV gene expressing immune cells, and as this is an accepted and safe duration for possible viremia rebound in previously ART controlled HIV-1 participants. Interruption of ART in previous clinical trials has led to a consistent re-emergence of viremia in approximately 2 weeks, with re-establishing a set point in 4-6 weeks (33). This approach has been proven to be safe for participants. Designed as an exploratory endpoint in our study, this duration of treatment interruption will allow determining whether participants' viral loads return to their pre-ART set points. Multiple clinical trials testing therapeutic vaccination for HIV-1 as a strategy for modifying the role of ART for controlling HIV-1 disease have used a similar interruption (formerly known as Structured, or Strategic, Treatment Interruption (STI)) for 12 weeks due to its safety profile. 2.4.1 ART Resumption Criteria Several safety parameters will be monitored and will be used to restart therapy earlier, if needed. These include:
1. CD 4+ T cell criteria: A decline of CD4+ count to less than 200 cells/mm3 in 2 consecutive measurements laboratory values obtained on different days, 2 weeks apart, or more than 50% drop in absolute CD4 count.  
2. Viral load criteria: ART will be initiated for any one of the following outcomes:
- viremia > 50,000 copies RNA/mL for four weeks
- viremia > 10,000 copies RNA/mL for 6 weeks
- viremia > 2000 copies RNA/ml for 12 weeks
- viremia > 400 copies/RNA for 24 weeks Operationally, this will allow for an acute viremic state that will need to resolve within 12 weeks, with high-level viremia only being allowed for a brief period. After the spike, the viral load should fall as the numbers of susceptible target cells are deleted, leaving just the transformed cells. Post-treatment control (defined as viremia <400 copies RNA/mL) should be achieved prior to Week 24 post-virus rebound. For any initial viral load greater than 400 copies/mL during weekly monitoring, participants must return within 3 business days for a repeat test.  
3. Clinical criteria: ART will also be re-initiated for any symptoms suggestive of a severe acute retroviral syndrome, particularly signs of altered mental status or viral meningitis (severe headache, neck stiffness), but including unexplained rash, lymphadenopathy, fevers, and occurrence of additional opportunistic infection (OI). ART will also be re-initiated if a participant tests positive for COVID-19 by PCR.  
4. If, in the opinion of the subject or the primary care physician, restart is desired for other reasons. It will be requested, but not required, to discuss this decision with the PI and research team beforehand, if feasible.
summary of changes
2.4.2 art withdrawal monitoring

Participants will be closely monitored weekly when they are off ART, and treatment can be restarted at the discretion of the participant, their HIV-1 physician, or the site PI. All patients on ART withdrawal will receive anti-OI prophylactic antibiotics. Despite a long history with the application of ATI for testing the therapeutic impact of candidate immunomodulatory treatment strategies, some studies have indicated that treatment interruptions, when devised with a medication sparing intent and without close clinical observation, may carry certain risks and are not always successful long-term (31). The host of published data on ATIs has recently been summarized in a Cochrane report (32). An important conclusion of this report is that ATIs continue to have applicability to the testing of new therapeutic concepts or drugs in well-characterized participant populations in the context of wellcontrolled clinical settings. However, ATIs without the addition of any other treatment modality, such as immunotherapy or with an ART sparing interval, are no longer supported. Based on recent trial results, the U.S. Department of Health and Human Services (DHHS) HIV-1 Treatment Guidelines (2015) state that, apart from unplanned ATI or short-term ATI due to drug toxicities, long-term ATIs are not recommended (34). The guidelines provide important guidance, advising that if therapy has to be discontinued, participants should be counseled about the need for close clinical and laboratory monitoring. They should also be aware of the risks of viral rebound, acute retroviral syndrome, increased risk for HIV-1 transmission, decline of CD4+ T-cell count, HIV-1 disease progression or death, development of minor HIV-1associated manifestations such as oral thrush, development of serious non-AIDS complications, development of drug resistance, and the need for chemoprophylaxis against opportunistic infections depending on the CD4+ T-cell count. Treatment interruptions possibly result in rapid reductions in CD4+ T-cell counts. The risks of repeated treatment interruptions are not clearly defined in the guidelines; however, data from the NIH's Strategies for Management of Antiretroviral Therapy (SMART) study suggest that cycles of treatment interruption at low CD4+ T cell counts could result in increased risk of death or disease progression. These risks continue to exist for short, prolonged, and repeated cycles of ATI. Participants in the current study will be monitored on a weekly basis during the 12week ATI and beyond. Participants will also be counseled with regard to the risks (outlined in the HIV-1 Treatment Guidelines) prior to ATI and weekly during ATI. During ATI, participants will be tested for COVID-19 if they experience symptoms consistent with COVID-19 and will restart ART COVID-19 positive by PCR. In addition, drug resistance data will be carefully reviewed by the investigator prior to allowing a participant to enter a second treatment interruption. Complications, such as acute retroviral syndrome when suspending ART or immune reconstitution syndrome when restarting ART, are more common in the setting of their *de novo* occurrence (i.e., when HIV-1 is first contracted and when ART is first initiated in the ART naïve patient). Even in these circumstances, their occurrence is rarely clinically significant with the potential exception of cryptococcal meningitis and reactivation mycobacterium tuberculosis disease in the case of immune reconstitution inflammatory syndrome. Irrespective, participants will be alerted to the potential for each of these respective events and instructed on alerting the study team of any sentinel symptoms so that clinical observation can be intensified. Additionally, participants with a history of mycobacterium tuberculosis latent or remote infection, cryptococcal meningitis, or clinically significant cardiovascular disease will be excluded from participating in the study without careful review by the protocol leadership and a more intensive discussion with the participant about the potential risks of reactivation or clinical manifestations of these respective conditions. Based on this background, treatment interruption following engraftment will be delayed until CD4+ T-cell counts recover to ≥ 300 cells/mm3 to provide a buffer above the 200 cell/mm3 threshold for restarting ART prior to the end of the prespecified 12-week period. Any clinically significant deterioration observed or reported during the treatment interruption including, but not limited to, signs of altered mental status or viral meningitis (severe headache, neck stiffness), the emergence of fever to greater than 38.5oC, severe lethargy, or signs/symptoms of a possible OI, any OI including esophageal thrush or pneumonia, or any deterioration that in the opinion of the study team or the participant's primary provider would place the participant at risk for significant morbidity will be monitored and will be used to restart therapy earlier, if needed. While understandably non-specific, the safety of the participant will be the primary consideration attending any clinical management criteria during the course of this clinical trial. Consideration for AMC-097 (Version 9.0) 07FEB2022 28 NCI Version Date *07FEB2022* CLIENT CONFIDENTIAL
reinstating an ATI once interrupted due to the side effects will only be considered after consultation with the FDA. The rationale for establishing a threshold for restarting ART prematurely if the CD4+ T-cell count goes below 200 cells/mm3 is based on the expectation that selective expansion of transformed cells is likely to occur in the setting of loss of non-transformed cells and an overall decline in total CD4 count. The anticipated risk of this strategy is expected to be mitigated by the limited duration of a low CD4 count accompanying high viremia. Clinical experience has demonstrated that the risk of OI occurrence increases over time. As an example, in the experience of the clinical investigators, unintentional interruption of *Pneumocystis carinii* pneumonia (PCP) prophylaxis is rarely associated with reactivation PCP sooner than several months. Additionally, while risk of OI occurrence is tightly associated with decline in CD4+ T-cell count, the level of viremia interacts with the rate of risk as well. Reinstitution of potent ART, which rapidly suppresses viremia within days to weeks in the setting of close clinical monitoring, will minimize the risk to the participant of significant clinical events. While an independent association of the level of viremia and risk of OI or other morbidities has not been established, the team will err on the side of caution and use the escalating viral load criteria described above as a threshold. This will be implemented more to be confident of the ability to control attendant inflammation and potential consequences, rather than suggesting that HIV-1 viremia is associated independently with increased risk of OI. Please refer to Appendix 1: App - 1C for the schedule of assessment for the ATI portion of the study, Appendix XII for the ATI disclosure form for participants, Appendix XI for ATI risk mitigation and counseling forms, and Appendix XIII for PrEP information sheets for participants.
summary of changes
2.4.3 post art withdrawal monitoring

Once ART is reinitiated, participants will be evaluated at Weeks 4 and 8 on ART. If viral load is not below the level of quantification by Week 8, participants will be evaluated every four weeks until the viral load is confirmed to be less than the level of quantification. A follow up visit will occur 24 weeks after resumption of ART. Routine safety studies, CD4+/CD8+ T cell counts, and viral load will be performed at Weeks 4, 8, and 24, and as needed between Week 8 and Week 24 to monitor safety and evaluate decrease of viral load to below level of quantification as described above. Plasma and PBMCs will be collected and cryopreserved at the time of ART resumption and at Weeks 4, 8 and 24 on ART. Please refer to Appendix 1: App - 1C for the schedule of assessment for the ATI portion of the study.  
2.4.4 Change in ART withdrawal criteria (protocol version 8)
The previous goal of ART withdrawal when T-cell count has risen to ≥ 500 cells/mm3 was not achievable for most participants. Some patients achieved a CD4 count of ≥ 300 cells/mm3. Resuming ART when CD4 is < 200 cells/mm3 is appropriate for safety reasons. Below 200, we expect participants to be prone to OIs, hence most providers use this as a threshold to begin prophylactic antibiotic. This change in the CD4 criteria will allow participants to begin controlled ART withdrawal earlier around 6 months to a year and not wait for two years to do so.  
ART withdrawal in these participants will provide critical information about the kinetics of the selection of HIV-resistant transplanted clones of CD4 T-cells and the potential kinetics of HIV reservoir decay in that setting. This will inform the design of future studies when models of the threshold for a potential requirement for a proportion for transformed cells required for cure might be identified. **In other words, the** information collected during a structured STI may provide sufficient information to model what fraction of the transplanted product should be HIV resistant stem cells in order to achieve an extinction of the HIV reservoir. Although it is not anticipated that any of the participants from earlier cohorts **will achieve control of their infection**
following an STI, we have observed an expansion of the transformed CD4 clone in participants **who became viremic due to ART non-compliance. Ultimately, the goal of** our treatment intervention is that the proportion of transformed CD4/HIV-resistance cells in recipients is sufficient such that they no longer need ART to control HIV infection. STI is the most certain way to establish that desired endpoint. Including participants from the earlier cohorts will assist in guiding the design of future clinical trials to achieve that goal.2.5 Correlative Studies 2.5.1 Biologic rationale and proposed mechanism of action for the study agent Survival in patients infected with HIV has improved dramatically with the advent of ART. However, neither a cure nor a vaccine exists. Infected individuals develop AIDS related malignancies such as lymphoma that are in general more aggressive and present with higher disease stages. Even after successful curative therapy of their malignancy, their survival depends on high compliance with ART therapy to prevent the relapse of lymphoma. Lack of immunological response despite virological suppression, issues with non-compliance or drug resistance, side effects of medications, and development of a well characterized but poorly understood chronic inflammatory status associated with different chronic illnesses are just a few of the complications that continue to negatively affect the quality of life and survival in patients with HIV related lymphomas. Hematopoietic stem cell (HSC) gene therapy for HIV may offer an alternative one-time treatment, with the possibility of controlling both the lymphoma as well as HIV itself by eliminating reservoirs of HIV that are responsible for persistence of the disease. Our proposed novel anti-HIV therapeutic candidate, autologous HSCs transduced with a triple combination of anti-HIV genes transferred by a single lentiviral vector is aimed at blocking HIV infection at different stages of the HIV life cycle. This approach provided strong pre-integration inhibition of HIV-1 infection, eliminated the generation of viral escape mutants, and prevented the integration of multiple, including resistant strains of HIV in various cell types in vitro. *In vivo* safety/efficacy studies in a humanized mouse model displayed excellent results including a selective survival advantage of anti-HIV gene modified cells and maintenance of normal human CD4+ cell levels. We also use a novel method of transduction that improves our transduction efficiency to a more clinically relevant level. Furthermore, for the first time in any HIV gene therapy approach, we are proposing to use a selectable surface marker on transduced cells to purify and transplant only anti-HIV vector transduced HSCs. Significantly, this improvement in transduction efficiency and selection for anti-HIV transduced HSC, resulted in decreased viremia in our model. We have successfully filed an application to the NIH Recombinant DNA Advisory Committee (RAC) and have been awarded an FDA IND approval and have started to manufacture the clinical GMP reagents for our clinical trial. We will be targeting HIV related lymphoma patients that require transplant as a part of their standard of care. This provides us an ethically acceptable setting to test the safety and efficacy of our product. By using a highly effective combinatorial anti-HIV gene therapy approach, developing a novel and significantly improved transduction process, revising transplant procedures to transduce HSCs before freezing and, most importantly, selecting and transplanting only the HIV resistant transduced HSCs, we expect to replace the patient's immune system with HIV resistant cells, thus depleting viral load in the absence of ART.
summary of changes
2.5.2 specific hypothesis

Anti-HIV gene expressing stem cells can safely reconstitute the hematopoietic system of the recipient and can produce a pool of mature myeloid and lymphoid cells that harbor anti-HIV genes capable of resisting HIV-1. As a secondary objective, we will interrogate two physical compartments of immune system, (i.e., peripheral blood (PB) cells and gut mucosa) for the presence, quantity and duration of gene modified HIV-1-resistant immune cells. In addition, we will determine the integration sites of vector sequences in circulating cells and monitor for any clonal expansion of hematopoietic cells.
summary of changes
2.5.3 relevant preclinical data

We have obtained strong preclinical data in both our *in vitro* and *in vivo* experiments demonstrating safety and efficacy of our tCD25 pre-selective combination anti-HIV lentiviral vector. This vector encodes a chimeric human/rhesus macaque TRIM5α isoform, a CCR5 shRNA, and a TAR decoy each of which inhibit HIV in a different stage of its life cycle. It also expresses a truncated/mutated form of human CD25 which is used as a selectable marker to purify transduced HSC away from untransduced HSC so an enriched population of HIV-resistant HSC can be transplanted into patients. SAFETY/FEASIBILITY/TOXICITY STUDIES: To evaluate the levels of enrichment of 1TAX vector transduced HPC, flow cytometry was performed on CD25 immunomagnetic bead purified cells. As displayed in Figure 2-B, enrichment levels ranged from 89.6% to 98.3% with an average of 94.2% (Post‐enrich). This was compared to initial transduction efficiencies, which ranged from 19.8% to 60.4% with an average of 33.0% (Pre‐enrich). Human HPC do not normally express CD25 and this is displayed as background expression, ~1.33% (Non-transduced, or NT). Flow through (ft) levels from the Stem Cell Technologies magnetic columns, ~2.07%, demonstrated that the majority of 1TAX vector transduced cells were collected during the enrichment procedure.  
Figure 2-B: Enrichment of 1TAX vector transduced cells: **Human CD34+ HPC were**  
  
transduced with the 1TAX vector, purified by CD25 immunomagnetic beads, and analyzed by flow cytometry for CD25 expression. Non‐ transduced cells (NT), flow through (ft)
Safety was observed in 1TAX vector transduction of HPC using colony forming unit assays and differentiation of these colonies into phenotypically normal macrophages. No significant differences in colony formation or expression of normal macrophage cell surface markers was observed in 1TAX vector transduced HPC compared to NT cells. Genetic stability of integrated 1TAX vector including the lack of deletions and rearrangements was also observed. The correct sequence of the integrated 1TAX vector was observed in transduced HPC. Expression of all three anti-HIV genes was detected in 1TAX vector transduced HPC. Potent down regulation of CCR5 expression was observed in 1TAX vector transduced cells (>94%, p<0.001) (Figure 2-C).  
Figure 1-C: Down regulation of CCR5 expression on 1TAX cells: Ghost‐**R5/X4/R3 cells**  
 transduced with the 1TAX vector were analyzed by flow cytometry for CCR5 cell surface expression. A representative histogram is displayed with NT cells (unshaded) and 1TAX vector transduced cells (shaded). Experiments were performed in at least triplicate. *** p<0.001 With the transduction of lentiviral vectors, there is the possibility for integration to occur in an area of the genome which may immortalize that specific transduced cell. Therefore, to determine if any immortalization occurred with 1TAX‐
transduced HPC, a long-term culture assay was performed similar to previously described (Modlich et al., 2009). No visual immortalization had occurred in either the 1TAX transduced or NT HPC cultures upon scoring 192 individual wells. To further evaluate the tumorigenicity of 1TAX vector transduced HSC, a NODRAG1-/-IL2rgamma-/- (NRG) knockout mouse model was used. Human CD34+
HSC were transduced with the 1TAX vector, enriched by CD25 immunomagnetic bead separation and transplanted into NRG pups (N=8). Engrafted mice were then left in the UC Davis vivarium for 6 months and transferred to the UC Davis Comparative Pathology Laboratory where they were euthanized and evaluated for any tumors. Gross necropsy and histologic examination of all major organs was performed. The anatomic pathologist and director of the comparative pathology lab confirmed that there were no tumors noted grossly or histologically. To evaluate the in vivo safety and toxicity of 1TAX vector transduced HSC, human CD34+ cells transduced with the 1TAX vector were transplanted into NRG pups (N=10). Twelve weeks post-transplantation, mice were screened for engraftment in the peripheral blood by flow cytometry. 1TAX transplanted HSC (55.5%) (N=10) displayed similar CD3+/CD4+ human T cell engraftment as NT HSC (51.6%) (N=7) transplanted mice. The phenotypic profile of peripheral blood 1TAX T cells was analyzed by multi-color flow cytometry. No significant differences in the percentage of the CD3+ T cell markers, human CD4+ (p=0.50), CD8+ (p=0.32), CD4+/CD8+ (p=0.34), CXCR4 (p=0.08), CD28 (p=0.17), and CD45RA+/CCR7+ (p=0.84), were observed when comparing NT and 1TAX vector transduced cells (Figure 2-D).  
Figure 2-D: Phenotypic profile of 1TAX vector transduced peripheral blood T cells: Human  
 CD34+ HSC were transduced with the 1TAX vector and transplanted into NRG mice. The peripheral blood of engrafted mice was analyzed by multi-parametric flow cytometry for T cell phenotypes NRG mice engrafted with 1TAX transduced and enriched cells were then evaluated for human immune cells in other hematopoietic sites such as the spleen, thymus, and bone marrow. 1TAX transplanted HSC (54.4%) (N=10) displayed similar CD3+/CD4+ human T cell engraftment levels as NT HSC (59.7%) (N=7) transplanted mice. No significant differences in the percentage of the CD3+ T cell markers, human CD4+ (p=0.36), CD8+ (p=0.64), CD4+/CD8+ (p=0.48), CXCR4
(p=0.20), CD28 (p=0.08), and CD45RA+/CCR7+ (p=0.62), were observed when comparing NT and 1TAX vector transduced cells. 1TAX vector transplanted HSC (41.7%) (N=10) displayed similar CD3+/CD4+ human T cell engraftment levels as NT HSC (42.9%) (N=7) transplanted mice. No significant differences in the percentage of the CD3+ T cell markers, human CD4+ (p=0.79), CD8+ (p=0.18), CD4+/CD8+ (p=0.13), CD3-/CD8+ (p=0.0.58), CXCR4 (p=0.09), CD28 (p=0.43), and CD45RA+/CCR7+ (p=0.34), were observed when comparing NT and 1TAX vector transduced cells. The levels of 1TAX B cells in the spleen and bone marrow and 1TAX macrophages in the bone marrow of engrafted NRG mice were also analyzed by flow cytometry. No significant differences in the levels of spleen B cells (p=0.47), bone marrow B cells (p=0.27), or bone marrow macrophages (p=0.22) were observed in 1TAX compared to NT cell engrafted mice. These results demonstrate that 1TAX vector transduced and enriched HSC are safe, are not toxic, and are able to successfully engraft in NRG and undergo normal multi-lineage hematopoiesis in the peripheral blood and lymphoid organs all while not causing tumorigenicity. EFFICACY STUDIES: The ultimate goal of HIV gene therapy is to transplant a HIV‐resistant immune system into HIV infected patients. To evaluate the efficacy of 1TAX vector transduced HSC in inhibiting HIV infection, both in vitro and in vivo challenge experiments were performed.  
First, an *in vitro* HIV-1 model system incorporating human CD34+ HPC derived macrophages was used. To evaluate the anti‐HIV efficacy of enriched 1TAX vector transduced cells, *in vitro* HPC derived macrophages were challenged with a CCR5‐
tropic BaL‐1 strain of HIV‐1. As displayed in Figure 2-E, significant resistance
(over 2 logs on days 14 and 21 post-infection) to HIV‐1 infection was observed in 1TAX vector transduced cells compared to NT cells (day 3 p=0.033, day 7 p<0.001, day 14 p<0.005, day 21 p<0.001, and day 28 p<0.001) as determined by p24 antigen Enzyme Linked Immunosorbent Assay (ELISA).  
Figure 2-E: **HIV‐1 challenge of 1TAX HSC derived macrophages: A) Nontransduced (NT)**  
 (**♦) and 1TAX vector transduced (■) HSC derived macrophages were challenged with a** CCR5-tropic BaL-1 strain of HIV-**1. On multiple days post‐infection, cell culture** supernatants were sampled and assessed for HIV-1 using a p24 antigen ELISA kit. Experiments were performed in triplicate. * p<0.05, ** p<0.01, *** p<0.001 An *in vitro* selective survival experiment was performed to determine if anti-HIV gene modified cells were capable of surviving during the course of an HIV-1 infection. In cultures containing a 1:1 and 2:1 ratio of combination vector transduced cells to untransduced cells, the pre‐infection Enhanced Green Fluorescent Protein (EGFP) percentage being, ~46% and ~73% respectively, increased to >84% EGFP positive cells by day 21 post‐infection for both BaL‐1 and NL4‐3. This increase was statistically significant (BaL‐ 1 50% p value=0.001, BaL‐
1 75% p value=0.05) (NL4‐3 50% p value=0.001, NL4‐3 75% p value=0.001).  
These results are in contrast to cultures containing EGFP‐alone control vector transduced cells where the EGFP percent positive cell population remained relatively constant on day 21 post‐infection (~42% and ~72% for ratios of 1:1 and 2:1, respectively) compared to the pre‐infection EGFP percentages of 46% and 70%
(BaL‐1 50% p value=0.23, BaL‐1 75% p value=0.69) (NL4‐3 50% p value=0.62, NL4‐3 75% p value=0.95).  
To determine if viral inhibition was occurring at the stages of pre-entry (due to the CCR5 shRNA) and post-entry/pre-integration (due to TRIM5α), cells challenged with both BaL-1 and NL4-3 were further analyzed by quantitative polymerase chain reaction (qPCR) for genomic HIV-1 provirus. A highly potent block of HIV-1 provirus formation was demonstrated. Anti-HIV vector transduced cells contained undetectable levels, similar to background levels in uninfected cells, of HIV-1 provirus as compared to control untransduced and EGFP-alone vector transduced infected cells at multiplicity of infections (MOIs) of 0.01 and 0.05 for BaL-1 and NL4-3 strains of HIV-1. These data confirm that the anti-HIV vector conferred a strong block to HIV-1 integration, therefore preventing HIV integration. The hallmark characteristics of progressive HIV infection include a decline in CD4+ cells and an increase in plasma viremia. To evaluate the in vivo anti‐HIV
efficacy of 1TAX transduced cells, a humanized NRG mouse model was used.  
NRG mice engrafted with 1TAX HSC were infected with either a CCR5‐ tropic BaL‐1 or CXCR4‐tropic NL43 strain of HIV‐1. On specific weeks post-infection, the peripheral blood of infected mice was analyzed by flow cytometry for CD4+ T cells and qPCR for plasma viremia. Mice engrafted with NT HSC (N=7) and infected with BaL‐1 displayed a depletion of CD4+ T cells from pre‐infection levels
(68.1%) to 15 weeks post‐infection (21.6%). This was in comparison and significantly different (p=0.0019) than mice engrafted with 1TAX HSC (N=7) and infected with BaL‐1 which did not display CD4+ T cell depletion with pre‐infection levels of 68.8% and 15 weeks post‐infection of 77.9% (Figure 2-F A). Similar results were observed in NT and 1TAX engrafted mice infected with NL43. NT
mice (N=4) displayed a decrease in CD4+ T cell levels from pre‐infection (78.1%)
to 15 weeks post‐infection (14.8%) and 1TAX mice (N=4) displayed a decrease in CD4+ T cell levels from pre‐infection (74.0%) to post‐infection (56.3%) (Figure 2-
F B). A significant (p=0.028) depletion of CD4+ T cells was observed on week 15 post‐infection when comparing NT and 1TAX mice. Even though a decrease in CD4+ T cells (pre‐infection to post‐infection) was observed in 1TAX mice infected with NL43, this decrease was not significant (p=0.23). These results demonstrate that maintenance of CD4+ T cell levels could be achieved when an enriched population of 1TAX‐transduced HSC is transplanted into NRG mice.  
Figure 2-F: Maintenance of human CD4+ T cells in vivo in 1TAX cell engrafted NRG mice  
 infected with HIV‐1: NRG mice engrafted with either untransduced (NT) or 1TAX vector transduced cells were infected intraperitoneally with a (A) CCR5‐tropic BaL‐1 or a (B) CXCR4‐tropic NL43 strain of HIV‐1. Various weeks post‐infection, mice were bled via tail vein and analyzed by flow cytometry for total human CD3+/CD4+ cell percent. Bar graphs display averages and standard deviations from seven mice per cohort for the B**aL‐1**
infections and four mice per cohort for the NL43 infections for CD3+/CD4+ cell levels pre-**infection and 15 weeks post‐infection. * p<0.05 and ** p<0.002**
Figure 2-G: Reduction in HIV-1 plasma viremia in NRG mice engrafted with 1TAX vector  
 transduced HSC: NRG mice engrafted with either untransduced (NT) or 1TAX vector transduced cells were infected intraperitoneally with a (A) CCR5‐tropic BaL‐1 or a (B) CXCR4-tropic NL43 strain of HIV‐1. Various weeks post-infection, mice were bled via tail vein and analyzed by qPCR for plasma viremia using a primer/probe set specific for the HIV LTR. Log RNA copies/mL for NT (**♦) (solid grey lines), 1TAX vector transduced (■) (solid** black lines), and background levels from uninfected (UI) mice (▲) (dashed grey lines). Line graphs display averages and standard deviations from seven mice per cohort for the BaL‐1 infections and four mice per cohort for the NL43 infections. * p<0.05 and ** p<0.005.  
Here, as we were able to initially enrich for 1TAX vector transduced cells prior to transplantation, we were able to generate a human immune system in the NRG mice which was resistant to HIV infection. This allowed for only a small percentage of NT and unprotected cells being transplanted with the majority of the transplanted cells protected. Combined, these results and preclinical data highlight the potential of our therapeutic candidate to succeed in our Phase I human clinical trial.  
2.5.4 Reason for selection of the assay methodology To evaluate the differentiation and persistence of anti-HIV HSC in recipient participants including functional and phenotypic changes in immune cell populations following HSC transplant, and to measure viral load and HIV response. Correlative studies, especially for gene therapy protocols, are critical to evaluate the feasibility, safety, and efficacy of the therapeutic intervention. The correlative studies can be divided into immunological and HIV studies, DNA monitoring studies, and monitoring for oncogenesis and hematopoietic clonality. We will evaluate various aspects of the anti-HIV lentiviral therapy including DNA monitoring, oncogenesis/clonality, and immunological studies. Long-term expression of anti-HIV genes and stability of the integrated vector will also be evaluated. The primary objectives of the immunology studies in this protocol are to monitor the reconstitution of the immune system, especially the homeostasis and function of T cell immunity in HIV-infected participants transplanted with antiHIV gene transduced stem cells. A large number of clinical trials have shown that chronic HIV infection results in depletion of key T cell subsets. 2.5.4.1 DNA monitoring studies The DNA monitoring core will evaluate engraftment (critical for efficacy) and safety of our approach in peripheral blood and gut tissue of transplanted participants. We will evaluate sites of vector integration, immune responses to foreign genes, potential silencing of anti-HIV genes (CCR5 shRNA, chimeric TRIM5α, and TAR decoy), and toxicity of the cells including tumorigenicity. Pre-transplant samples will be collected from the UC Davis GMP facility and also from leftover infusion bags. Post-transplant, both peripheral blood and biopsies will be obtained according to the timeline in Appendix I.  
Approximately 20 mL of ethylenediaminetetraacetic acid (EDTA) anticoagulated blood specimens will be collected for DNA monitoring studies
(see Appendix VII for additional details of DNA monitoring studies).  
Objective 1 Determine the levels of *in vivo* gene marking and vector copy number in 1TAX vector transduced cells.
Total DNA will be extracted from the peripheral blood of participants transplanted with the 1TAX vector transduced cells. qPCR will be performed with a vector-specific primer/probe set specific for the chimeric TRIM5α transgene. The levels of glyceraldehyde-6-phosphate (GAPDH) expression will also be evaluated as an internal control.  
Objective 2 Determine integrated 1TAX vector sequence and stability.
To evaluate integrated 1TAX vector sequence and stability, sequencing will be performed. Total DNA will be extracted from the participant's peripheral blood. PCR amplification and sequencing (with vector specific primers) of integrated 1TAX vector will be performed. Sequences obtained will be evaluated for rearrangements, deletions, and additions.  
Objective 3 Evaluate expression of the anti-HIV genes:
To determine if all three anti-HIV genes (CCR5 shRNA, chimeric TRIM5α, and TAR decoy) are being expressed long-term, qPCR will be performed. Total RNA will be extracted from the participant's peripheral blood. qPCR with gene specific primers will be performed to determine expression levels. GAPDH expression levels will be used as an internal control.  
Objective 4 Evaluate clonality of 1TAX transduced cells in duodenal tissue:
In any gene therapy protocol, there is a risk of insertional oncogenesis due to random insertion of the vector. We will routinely monitor participants for any evidence of clonality. This is important for stem cell transplantations in lymphoma patients since there is an inherent risk of secondary malignancies (about 5-8%) due to the chemotherapy both as a treatment for controlling the malignancy and as a transplant preparative regimen. We will perform LAM-PCR every 6 months for 1TAX vector integrant sites and look for evidence of increasing oligoclonality or monoclonality, defined as 2-fold increase over two successive timepoints to > 20% of total transduced cells.  
Objective 5 Determine if an immune response develops in participants due to the expression of the chimeric TRIM5α transgene.
The chimeric TRIM5α anti-HIV gene contains a 13-amino acid patch which is foreign to native human TRIM5α. Therefore, it is possible for an antibody based immune reaction to occur against cells expressing this region. However, this is highly unlikely in a myeloablative setting when immune tolerance can be induced against more prominent antigens such as human leukocyte antigen (HLA). Also, in our preliminary data, no stimulation was observed in cells pulsed with this amino acid region. Still and in accordance with the FDA, we will monitor participant serum for any antibody production by ELISA.
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2.5.4.2 immunological studies

The immunological studies will be done in 2 compartments: peripheral blood and gut mucosa. A large number of clinical studies have demonstrated that chronic HIV infection results in preferential depletion of key T cell subsets, namely naïve and various T memory cells. An increased level of T cell activation has also been demonstrated in association with disease pathogenesis in AIDS patients as measured by HLA-DR and CD38 co-expression on CD8+ T cells. More recently, other cell surface markers have also been tested including CD69 and CD71. In addition to the imbalanced Th1/Th2 subsets, HIV infection also induces impairment in Th17 and regulatory T cells (Treg), an imbalance that reflects a systemic immunologic defect more pronounced in advanced HIV disease and after effective viral suppression with ART. In the current proposal, homeostasis and differentiation of major T cell populations, and HIV-specific T cell responses will evaluate the adaptive antiviral T cell functions. Furthermore, Type-1 and Type-2 T cell responses to common pathogens including CMV and EBV antigens will be assessed. Additional components of the innate immune system, including NK, NK-T cells, and dendritic cells will be examined in order to characterize functional immunologic changes following transplant. Furthermore, antigen-specific T cell responses will evaluate the adaptive antiviral T cell functions. Approximately 40 mL EDTA anti-coagulated blood specimens and 5 mL of non-anticoagulated blood (serum) will be collected for immunological studies. Out of the 40mL blood, about 2-mL whole blood will be used as fresh for phenotyping assays to determine the absolute counts of T cells, B cells and NK cells. The rest of blood will be:
- Isolate plasma and peripheral blood mononuclear cells (PBMC) (38 mL):  
o 10 mL plasma will be collected and stored in 1 mL aliquots for cytokine assays in later days (Year 3 and Year 4).
o 10-20x106 cells will be used in assays immediately as fresh (listed as Functional assay in Section 2.5). Out of these cells, a minimum of 5x106 PBMC will be used for phenotyping assays of immune cell populations. A minimum of 10x106 PBMC will be needed for T cell function assays (T cell responses to pooled HIV peptides stimulations, to CEF (CMV-EBV-influenza virus), as well as culture median only (negative control) and to one of the positive mitogenic stimulations (positive controls, PMA or SEB (Staphylococcal enterotoxin-B)).  
o The rest of PBMC will be cryopreserved in 10% DMSO-FBS and stored in liquid nitrogen for testing in a later time, tentatively scheduled in Year 3 and Year 4. The expected number of PBMC in cryopreservation is 20-30x106, but could be less, especially for earlier time points post-transplant. In this case, we will make sure at least 5x106 cells will be preserved for any possible need of retesting.
From the plasma (10 mL) and serum samples (3 mL), aliquot as stored samples till batched assays, tentatively scheduled for Year 3 and Year 4. Cytokines studies will focus on the increased inflammatory status seen in patients with controlled HIV associated with immune exhaustion. They include but not limited to TNF-alpha, interferon gamma, IL2, IL10 and also other markers of chronic immune activation/exhaustion such as soluble PDL1/2.
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reconstitution of immune system in

Our laboratory will use established polychromatic flow cytometry panels to enumerate lymphocyte subsets and antigen specific cytokine expression in CD4 and CD8 T cells. Isolated PBMC from 40 mL EDTA anti-coagulated blood specimens will be collected from each participant at baseline, and at 3, 6, 12, and 24 months post HSCT for the following studies. The processing for participants enrolled at UC Davis will be done at UC Davis Medical Center. Critical assays will be performed with fresh whole blood and freshly isolated PBMC. Additional PBMCs will be stored in liquid nitrogen for later time assays (and necessary re-test).
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- chimerism of engrafted transformed

A portion of whole blood sample (~5mL) and a minimum of 10 x 106 PBMC will be tested in the flow cytometric assays to:  
a. Determine the proportion of circulating chimeric CD34+ cells expressing CD25.  
b. Determine the proportion of circulating chimeric non-lymphoid immune cells expressing CD25 will be explored employing a variety of antibody subsets to identify the most robust subset believed to reflect engrafted and persistent transformed populations.  
c. Consideration will be given to sorting these populations for PCR
verification of TRIM5ɑ genotype.  
- Mixed lymphocyte reactions:
In patients who are losing engraftment of the transduced cells, mixed lymphocyte reactions will be monitored in order to determine whether unmodified, post-SCT PBMC recognize and kill engrafted, modified cells. A mixed lymphocyte reaction will be performed using post-SCT PBMCs obtained when more than 50% drop in transduced cell chimerism is determined by PCR. PBMC obtained from 20 cc of peripheral blood will be mixed with pre-SCT modified stem cells
(generated from remaining CD34+ depleted CD4+ T cells from preSCT leukapheresis). This will be performed in the immunology core.  
Gene modified cell killing and markers of T and NK cell activation and function will be determined in the ex vivo experiments using flow cytometric analysis. To study the homeostasis of T Lymphocytes and their subsets, a small portion of whole blood sample (~2-mL) and a minimum of 5x106 PBMC
will be tested in the flow cytometric assays to:  
a. Determine memory and naïve CD4 and CD8 T cells and T cell activation:
A multi-parameter test will be performed to detect viable CD3, CD4, CD8, CCR7, CD45RA, CD38, HLA-DR, CD27, CD57, Ki67 and viable cell stain. Naïve, central memory and effector T cells will be analyzed by expression of CCR7/CD45RA on T cells that are gated on viable-cell/CD3 and CD4 or CD8 staining. T cell activation will be measured using combinations of CD38/HLA-DR as well as CD27, CD57 and/orKi67.  
b. Determine the Treg, NK, NK-T cells, and DC subsets:
These will be examined with antibodies to CD45, CD11c, CD123, CD3, CD4, CD8, CD25, CD16, CD56, IL-7R, FoxP3, and viable cell stain. Expression of CD25+/ FoxP3+/IL-7R- will be used to identify Treg in viable CD3/CD4 cells. NK and NK-T cells will be identified by markers of CD3, CD16/CD56, and CD8. Expression of homing molecules (integrin α4β7, CD62L, CCR9, CD18, CD31 and CD54) and inhibitory and activating receptors on NK cells will also be included in this panel pending on specimen availability.  
c. Pending specimen availability, homeostasis of B cell classes and expression of T cell homing molecules (integrin α4β7, CD62L, CCR9, CD18, CD31, and CD54) will also be accessed (at baseline and month 12).
- Functional responses upon reconstitution of T Cell Immunity Isolated PBMC will be cultured with mitogen or HIV-specific antigens and used in the following studies. For participants enrolled at UC Davis, the processing starts as soon as possible after blood collection, usually within 1-hour. For participants enrolled at other sites: EDTA blood samples will be sent to UC Davis with chilled packaging via FedEx Priority Overnight shipping: a. Total Th1/Th2/Th17 subsets upon mitogen stimulation:
The detection panel will be: viable stain, CD3, CD4, CD8, and intracellular Granzyme-B, IFNγ, TNFα, IL2, IL4, IL10, and IL17a.  
Th1/Th2/Th17 subsets will be identified as IFNγ/TNFα/IL2, IL4/IL10, or IL17a, respectively, on viable CD4 T cells.
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b. hiv antigen specific t

PBMC stimulated with HIV-gag peptides will be examined with a panel of viability stain, CD3, CD4, CD8, and intra-cellular IFNγ, TNFα, IL2, IL4 and IL17a. The profile of cytokine(s) produced in viable CD4 or CD8 T cells will be analyzed and correlated to Granzyme- positive T cells. T cell responses to Staphylococcal enterotoxin-B (SEB) will be used as positive controls. Another control antigen, CMV-EBV-influenza virus (CEF) peptides antigens will be used as an indicator of T cell responses to pathogens common to human participants.  
c. T cell proliferation responses to HIV gag stimulations:
If additional PBMC are available (15x106 cells more), proliferation responses will also be tested using CFSE (Carboxyfluorescein succinimidyl ester) assays. Furthermore, at one-year post transplant, based on the American Society of Blood and Marrow Transplantation (ASBMT) guidelines, participants will be immunized with inactivated vaccines. Acquisition of immunity against a neoantigen can therefore be assessed in these patients by their response to immunization with a replication incompetent antigen such as tetanus toxoid.  
Analysis of the extra-vascular compartment of the immune system (gut mucosa: Immune restoration in the duodenum is profoundly blunted with absolute numbers of CD4+ T cells in the lamina propria changing little after prolonged viral suppression. We believe that a thorough evaluation of immune effects of our product will be enhanced by demonstration of transduced cells in duodenal tissue. It is anticipated that transduced cells will home to the lamina propria and will be particularly enriched during HIV treatment interruption. Presence of transduced immune cells in gut associated lymphoid tissue (GALT) will likely be a hallmark of the success for our gene therapy protocol. Our HIV team members are leaders in the area of gut immunity and reconstitution in response to HIV infection, with a long track record of performing upper endoscopy safely in HIV research participants, having performed over 100 procedures on 60 research participants over the last 4 years. To demonstrate homing/persistence of transduced lymphocytes in this compartment, the distal duodenum will be sampled at 6 months posttransplant (or when CD4 count reaches above 300 cell/mm3) and will be repeated two months after the first GI biopsy. This will facilitate addressing two important questions, namely the uptake of the transduced population of lymphocytes into the extravascular compartment and the degree of enrichment that occurs following selective survival pressure on these lymphocytes during ART withholding. The categorical residence of transduced cells and both their functional integrity and that of the local immune system in general will be assessed for both IHC and digestion into single cell suspension for flow cytometry. The distal duodenum will be sampled by a designated gastrointestinal (GI) physician at each center at 6 months post-transplant (or when CD4 count reaches above 300 cell/mm3) and will be repeated two months after the first GI biopsy. This will be under separate consent form for the study and the participation therefore is not mandatory for enrollment in the main study (see Appendix V).  
- Tissue biopsies will be immediately processed at the time of endoscopy for cryopreservation and for both subsequent IHC and digestion into single cell suspension for flow cytometry. Cryopreserved samples can be used for HIV quantitation and sequencing, RNA microarray, and quantitation of transduced cells by PCR of the inserted vector.  
- IHC will be performed to enumerate critical populations of the mucosal immune system, specifically CD4+ and CD8+ T cells and T-regulatory cells.  
- Flow cytometry will be performed in unstimulated CD4+ and CD8+ T
cells and T-regulatory cells.  
- Functional assays of immune function will be performed by incubating duodenal cells with mock challenge; SEB, CEF, and HIV gag peptides for intracellular cytokine measurement by flow cytometry of TNFα, IFN-γ, IL-2, IL-17a, and CD103.
These experiments are expected to demonstrate both the persistence of the transduced lymphocyte population, as well as improved immunologic function in this compartment, which can be compared to the time series of parallel immune function assays performed on PBMCs over the same time period.  
2.5.4.3 Virologic studies A. ART resistance and co-receptor usage: These studies will be performed at baseline, 2 and 10 weeks and then 6, 12, and 24 months posttransplant.  
B. To characterize changes in the HIV reservoir and viral diversity:
Additional blood samples will be collected at baseline, 2 and 10 weeks and then 6, 12- and 24-months post-transplant for HIV reservoir characterization. These studies will include quantitative PCR analysis of PBMC-associated HIV DNA, RNA (including unspliced and multiply spliced forms) and 2-LTR circles. In addition, single genome sequencing of HIV gp120 and HIV pol will be performed to understand sequence evolution following transplantation and detection of minority resistance variants. Single-copy HIV from plasma will also be determined at each of these time points. Samples will be sent to the Timothy Henrich Laboratory at University of California, San Francisco.
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2.5.5 1tax lentiviral vector stability

The marker is a stably integrated lentiviral vector harboring a combination of antiHIV genes and a pre-selective marker gene. As the 1TAX vector is stably integrated, it will remain permanently in the transduced cells.  
2.5.6 Technical performance characteristics, positive and negative controls, and methods of scoring for the assays
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study t lymphocytes and their

The flow cytometry data is highly reproducible and accurate. Variability will be observed from participant to participant; however, samples will be evaluated and compared from the same participant to decrease variability. Flow cytometry positive controls will be obtained from normal healthy donors. Negative controls are isotype stained cells.  
The flow cytometry data will be scored by percent + cell populations staining for the specific immune cell surface markers. Functional responses upon reconstitution of T cell immunity The functional cytokine secretion data is highly reproducible and accurate. Variability will be observed from participant to participant; however, samples will be evaluated and compared from the same participant to decrease variability. The functional cytokine secretion positive controls will be obtained from normal healthy donors. Negative controls are unstimulated cells from the same participant. The functional cytokine secretion data will be scored based on the levels of cytokines as measured in ng/mL. Monitoring for insertional oncogenesis and clonality of hematopoiesis after gene therapy We will use LAM-PCR (Linear Amplification Mediated PCR), a highly accurate method that is able to specifically identify the sites of integration of the 1TAX vectors. Variability will be observed even in the same participant samples as the vector is capable of integrating in 1000s of places in the targeted genome. There are no positive or negative controls for this assay. This is specific for 1TAX vector integration sites. It is not possible to specifically integrate a 1TAX lentiviral vector into the chromosome of a host genome. Insertional oncogenesis and clonality will be scored based on the sites of integration of the 1TAX vector and the number of events correlating to each specific integration site. DNA monitoring studies for in vivo gene marking, vector stability, and anti-HIV gene expression These assays are highly accurate and specific due to the use of primers specific for the 1TAX vector and expressed genes. Variability will be observed between participant to participant due to the different levels of engraftment. This will affect the levels of *in vivo* gene marking and anti-HIV gene expression. Variability will be minimized by comparing the results of these assays within each sample from the same participant. 1TAX vector stability should be consistent as we have not observed any deletions or rearrangements in our preclinical data. The positive control for *in vivo* gene marking is the transfer plasmid used for vector production, which includes the psi sequence. The negative control is a plasmid that does not contain the vector specific psi sequence. 1TAX vector stability positive control is again the transfer plasmid used for vector production. The negative control for 1TAX vector stability is genomic DNA from untransduced cells. The positive control for anti-HIV gene expression is total RNA from 1TAX vector transduced HEK-293T cells. The negative control for anti-HIV gene expression is total RNA from untransduced cells. In vivo gene marking will be scored by 1TAX vector copies per cell. Vector stability will be scored based on the correct size of the PCR product in comparison to the positive control transfer plasmid. Anti-HIV gene expression will be scored by relative gene expression as compared to GAPDH expression (housekeeping gene).
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monitoring for immune response to

The reproducibility of this assay will vary from participant to participant. This assay is designed to detect any immune response which may occur with the expression of the chimeric TRIM5α transgene and so we may never detect any immune response in participants. Accuracy will be assessed by baseline plasma levels obtained from participants prior to transplantation. The positive control for the TRIM5α ELISA is an anti-chimeric TRIM5α antibody generated in rabbits. The negative control is plasma from a normal healthy donor. The immune response to the chimeric TRIM5α will be scored based on the levels of anti-chimeric TRIM5α antibody in ng/mL. Monitoring of transduction efficiency and stem cell capabilities of transduced hematopoietic stem cells in immunocompromised mouse models Transduction efficiencies have been consistently reproduced to exceed 50% of the cell population. Variability is expected from participant to participant sample as we have observed in our preclinical data. The positive control for transduction efficiency is transduced HEK-293T cells. The negative control is an isotype control for flow cytometry. Transduction efficiency will be scored based on CD25+ cell expression by flow cytometry.  
2.5.7 The site performing the correlative studies UC Davis will perform the correlative studies. Dr. Pollard's lab will perform the immunological core studies and Dr. Anderson's lab will perform the DNA monitoring core studies. Specialized immunologic and virologic assays will also be performed in the laboratory of Dr. Timothy Henrich at University of California, San Francisco. Basic immunology studies will be performed according to the standard of care by CLIA-certified local laboratories at each participating institution.  
2.5.8 Additional sample for safety investigation
In the unusual circumstance where an unexpected or sudden change in the subject's clinical status occurs where product safety concerns raise a relevant question requiring additional blood samples outside the specified time points, participants may be asked for an additional blood sample (maximum of 30 mL of EDTA whole blood in a purple top tube), at the local PI's discretion in consultation with the protocol chair. The samples will be shipped to Dr. Pollard's Laboratory at UC Davis (see Appendix VII), and will be analyzed per the protocol correlative studies AMC-097 (Version 9.0) 07FEB2022 47 NCI Version Date *07FEB2022* CLIENT CONFIDENTIAL
relevant for the safety concern, such as mixed lymphocyte reactions. It is not anticipated that this will occur with every subject or on more than one occasion outside of the current schedule of events.
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3.0 participant selection

A rostered AMC investigator must document that each protocol participant meets meet all stated eligibility criteria. Participating sites must have documentation that each eligibility requirement is satisfied prior to participant enrollment. In compliance with CTEP policy, no exceptions to eligibility criteria will be granted under any circumstance.  
General participant population eligible for this trial: Male or female participants greater than 18 years of age with HIV-1 related lymphoma that are refractory/resistant to first line of therapy, or have relapsed after achieving a remission, and are eligible for an autologous HSCT as a part of their routine therapy are eligible for this study. The type of the chemotherapy and the number of cycles prior to the study, as well as the timing of stem cell collection in relation to the last chemotherapy, will be determined based on the transplant physician discretion and each individual institution guidelines. **There**
should be a minimum of 2 weeks from the last day of any chemotherapy (except the one used for stem cell mobilization) before a participant can be enrolled on this trial. While screening studies may be performed within 8 weeks of the stem cell collection, enrollment in the screening segment should occur before stem cell collection and no more than 1 week prior to stem cell collection.  
NOTE: Institutions may use this section of the protocol as an eligibility checklist for source documentation if it has been reviewed, signed, and dated before registration by the study investigator. If used as source documentation, this checklist must be printed, the investigator must check each item to document their assessment that the participant meets each eligibility criterion, and the completed checklist must be maintained in the participant's chart. All questions regarding eligibility should be directed to the study chair. Participant ID Number: 097 - ___ ___ ___ - ___ ___ ___ Patient's Initials (L, F, M): ___ ___ ___
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3.1 eligibility criteria

3.1.1 Inclusion criteria associated with type and status of lymphoma, one of the following must be applicable:  
____ 3.1.1.1 Biopsy-proven intermediate or high-grade non-Hodgkin's lymphoma, meeting one of the following criteria (timeline 8 months prior to enrollment in the screening segment*):
- In partial remission,
- Relapsed after initial complete remission,
- Failed induction therapy, but responds to salvage therapy (i.e.,
chemosensitive disease),  
- In complete remission with high-risk features as specified by the International Prognostic Index (See Appendix VIII).  
____ 3.1.1.2 Biopsy-proven advanced stage follicular lymphoma, that have failed at least two lines of therapy multi-agent chemotherapy, but responds to
salvage therapy (i.e., chemosensitive disease) (timeline 8 months prior to enrollment in the screening segment*).  
____ 3.1.1.3 Biopsy-proven advanced stage Mantle cell lymphoma with Ki-67 > 10%
in first complete remission (timeline 8 months prior to enrollment in the screening segment*).  
____ 3.1.1.4 Biopsy-proven Hodgkin's lymphoma, meeting one of the following criteria (timeline 8 months prior to enrollment*).  
- In first, or greater relapse after initial complete remission, - In partial remission, - Failed induction therapy, but responds to salvage therapy (i.e.,
chemosensitive disease).  
____ 3.1.1.5 Biopsy-proven Burkitt's lymphoma, meeting one of the following criteria
(timeline 8 months prior to enrollment*):
- In second complete remission after relapse following initial complete remission,
- Failed induction therapy, but responds (very good partial remission, complete remission, or near complete remission) to salvage therapy (i.e., chemosensitive disease).  
____ 3.1.1.6 Biopsy proven plasmablastic lymphomas, or peripheral T cell lymphoma
(with the exception of ALK+ type in first or second complete remission) (timeline 8 months prior to enrollment*).
***NOTE**: Patients meeting the following criteria are exempt from the 8month timeline and do not require additional biopsy:  
- Patients who have never achieved a complete remission on the last biopsy-proven site of disease and went on to the next therapy then achieved a complete remission.  
- Patients who relapsed quickly (within 3 months of their last chemotherapy) and now have achieved a complete remission with salvage therapy.  
3.1.2 Inclusion criteria associated with HIV-1 status
____ 3.1.2.1 HIV-1 infection, as documented by any federally approved, licensed HIV
rapid test performed in conjunction with screening (or ELISA, test kit, and confirmed by Western blot or other approved test). Alternatively, this documentation may include a record demonstrating that another physician has documented the participant's HIV status based on either: 1) approved diagnostic tests, or 2) the referring physician's written record that HIV infection was documented, with supporting information on the participant's relevant medical history and/or current management of HIV infection.
____ 3.1.2.2 Must be on a multi-drug anti-HIV regimen (excluding zidovudine [AZT,
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ZDV, Retrovir®, or agents containing zidovudine (e.g., Combivir® and Trizivir®)], and efavirenz [Sustiva®, or agents containing efavirenz (e.g., Atripla®)]).  
Participants on zidovudine [AZT, ZDV, Retrovir®; including Combivir®
and Trizivir®] and efazirenz [Sustiva®; including Atripla®] must switch to an alternative regimen without anticipated drug-drug interactions or myelosuppressive properties based on known viral resistance patterns and/or ART history, such as raltegravir and Truvada (emtricitabine and tenofovir) at least two weeks prior to the transplant.  
_____ 3.1.2.3 Participant taking ARTs must satisfy one of the following:  
- Undetectable HIV viral load (< 50 copies/mL). For patients who have had negative viral loads in the past 6 months and no known HIV viral load >500 copies/mL within the last 6 months, minor fluctuations of viral load (isolated escalations up to 500 copies/mL) are acceptable. The participant's history of negative viral loads may be documented with recent laboratory results and/or a record from the participant's HIV care provider.  
- If viral load is detectable at < 2000 copies/mL a review of previous antiretroviral regimens or previous genotypic or phenotypic testing which indicate the ability to fully suppress virus by addition of sensitive drugs must be performed. This review will be carried out by the protocol ID team or the ID specialist caring for the patient.  
- If viral load is detectable at ≥ 2000 copies/mL, a current HIV genotype and/or phenotype must be obtained. If a HAART regimen to which the patient's virus is sensitive can be determined based on genotype and previous antiretroviral experience, then the patient will be considered eligible in this regard. This review will be carried out by the protocol ID team or the ID specialist caring for the patient.
3.1.3 General Inclusion Criteria (timeline: within 8 weeks prior to enrollment in the screening segment, unless otherwise specified)  
____ 3.1.3.1 Karnofsky performance status of 70-100%. ECOG performance status <
[2] (see Appendix II)
____ 3.1.3.2 SGOT and SGPT ≤ 2.5 times upper limit of normal (ULN). Serum bilirubin ≤ 2.5 times ULN except for participants who are on atazanavir or indinavir, or with elevated indirect bilirubin related to bilirubin conjugation issues such as Gilbert's disease, provided that the participant's direct bilirubin is within normal institutional limits.  
____ 3.1.3.3 Participants who are hepatitis C virus antibody positive, or hepatitis B
virus surface antigen positive must be free of clinical evidence of cirrhosis as determined by the principal investigator in consultation with the institutional Gastroenterology Service. Timeline: within 3 weeks prior to enrollment.  
____ 3.1.3.4 Participants with Hepatitis B should be on appropriate anti-viral therapy at the time of the transplant, and their viral load should be negative. Timeline: within 3 weeks prior to enrollment.  
____ 3.1.3.5 Serum creatinine ≤ 2 times ULN. Timeline: within 3 weeks prior to enrollment.  
____ 3.1.3.6 Creatinine clearance ≥ 60 mL/min by the modified Cockcroft-Gault Formula. Timeline: within 3 weeks prior to enrollment.  
____ 3.1.3.7 PT/PTT ≤ 2 times upper limit of normal (ULN), or international normalized ratio (INR)/PTT ≤ 2 times the ULN. Timeline: within 3 weeks prior to enrollment.  
____ 3.1.3.8 FEV-1 or DLCO (corrected for hemoglobin) ≥ 50% predicted. Timeline:
within 4 weeks prior to enrollment.  
____ 3.1.3.9 LVEF ≥ 50% by 2D ECHO or MUGA scan. Timeline: within 4 weeks prior to enrollment.  
____ 3.1.3.10 Not pregnant or nursing, with negative serum pregnancy test. Pregnant women are excluded from this study because the conditioning regimen has the potential for teratogenic or abortifacient effects. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with BEAM, breastfeeding should be discontinued. These potential risks may also apply to other agents used in this study. Timeline: within 3 weeks prior to enrollment.  
____ 3.1.3.11 Participants should agree to practice effective contraceptive precautions and to use at least one method of contraception for the duration of the study and for 3 months post-transplant.  
____ 3.1.3.12 Age ≥18 years. Because only adult transplant centers are participating as study sites.
____ 3.1.3.13 Life expectancy of greater than 3 months. ____ 3.1.3.14 Ability to understand and the willingness to sign a written informed consent document.  
____ 3.1.3.15 Receipt of a stable ART regimen for at least 3 weeks prior to enrollment.
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3.2 exclusion criteria

Participants who do not fulfill the criteria as listed in Section 3.1 above, are ineligible.  
Additionally, the presence of any of the following conditions will exclude a participant from study enrollment (timeline for all the exclusion criteria is within 8 weeks prior to enrollment in the screening segment):  
____ 3.2.1 Participants with > 5% involvement of bone marrow by malignant cells (either by manual count or flow cytometry) prior to stem cell collection.  
____ 3.2.2 Participants with any abnormal cytogenetics in the bone marrow not related to the lymphoma, and not deemed to be constitutional.
____ 3.2.3 Participants with unexplained anemia and/or thrombocytopenia.  
AMC-097 (Version 9.0) 07FEB2022 52 NCI Version Date *07FEB2022* CLIENT CONFIDENTIAL  
____ 3.2.4 Participants with clear evidence of myeloproliferative disorders, or myelodysplastic disorders in the marrow.  
____ 3.2.5 Presence of any active CNS disease at the time of evaluation (parenchymal or leptomeningeal).  
____ 3.2.6 Any history of HIV-1 associated encephalopathy. ____ 3.2.7 Participants with persistently low CD4 counts less than 200 and a history of any AIDS-defining infection in the last 6 months before screening are excluded from the study.  
____ 3.2.11 Intractable, severe diarrhea, defined as > 1.500 cc diarrheal fluid per day, or diarrhea causing persistent severe electrolyte abnormalities, or hypoalbuminemia.  
____ 3.2.12 History of active myocardial ischemia, cardiomyopathy, uncontrolled dysrhythmia, or congestive heart failure within the last 6 months before enrollment.  
____ 3.2.13 Dementia of any kind. ____ 3.2.15 History of Grade III hemorrhagic cystitis due to prior cyclophosphamide chemotherapy.  
____ 3.2.16 History of other prior malignancy, except squamous cell carcinoma of the cervix or anus, superficial basal cell or squamous cell skin cancer, or other malignancy curatively treated more than 5 years ago before enrollment.  
____ 3.2.17 Active psychosocial condition that would hinder study compliance and follow-up. ____ 3.2.18 Any perceived inability to directly (and without the means of a legal guardian)
provide informed consent.  
____ 3.2.19 Any medical or physical contraindication, or other inability to undergo HPC
collection.  
____ 3.2.21 Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements.
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3.3 number of participants to

____ 3.2.9 Active CMV retinitis, or other active CMV-related organ dysfunction.  
____ 3.2.10 Relapse of pneumocystis carinii pneumonia within the past year before enrollment.  
____ 3.2.14 Seizures within the past 12 months before enrollment. ____ 3.2.20 Participants who are receiving any other investigational agents. (Optional unless this section is used as an eligibility checklist)
This study will enroll a minimum of 2 participants and a maximum of 18 AMC-097 (Version 9.0) 07FEB2022 53 NCI Version Date *07FEB2022* CLIENT CONFIDENTIAL
____ 3.2.8 Symptomatic/active bacterial, or fungal, or any other opportunistic infection. Physician Signature: ______________________________ Date: ___________________
3.3.2 Accrual rate Approximately 1 participant per month.
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3.4 recruitment methods

Participants will be recruited from UC Davis, AIDS Malignancy Consortium (AMC) sites as well as from other referring clinics (throughout California and beyond) and will be enrolled and treated on protocol at either UC Davis or AMC sites. Recruitment is open to all minorities and both genders. Although distributions may vary by disease type, our recruitment procedures have been developed to enroll participants who are representative of the respective target population. Screening of participants will be done by PIs as well as other members of the transplant team at either institution. All procedures will be performed at the UC Davis Cancer Center, UCSF Adult Hematology and BMT Clinic, the UCSD Moores Cancer Center, and Memorial Sloan Kettering Cancer Center. The UC Davis IRB, UCSF Committee on Human Research, the UCSD Human Research Protections Program, and the Memorial Sloan Kettering Cancer Center Institutional Review Board/Privacy Board will oversee study conduct at their respective sites. After the first 3 participants are recruited to the protocol and dependent on the pace of participant recruitment to the protocol, a decision will be made by principal investigator and in consultation with AMC Steering Committee to extend the recruitment to other AMC sites. Since it would not be possible to contact all of the participants associated with the study to obtain consent, the lead site (UC Davis) is requesting a waiver of HIPAA Authorization for recruitment purposes only. The PIs/research team will identify potential participants while at clinical appointments and access medical records to verify eligibility prior to the treating physician and research coordinator approaching the potential participant. Consent to participate and HIPAA Authorization to access additional information in the medical records will be obtained prior to enrollment into the study. For a description of the Cancer Center's plan to safeguard the Protected Health Information (PHI) from improper use and disclosure as well as assurance that the PHI will not be inappropriately reused or disclosed to any other person or entity (See Section 11.3). All the information disseminated to participants (handouts, brochures, etc.) and any advertisements will be first approved by the IRB for the site. Screening requirements outlined in Section 7.1 and Appendix I will be used to meet inclusion or exclusion criteria that are defined for the study (see Section 3.1 and 3.2). Participants will be informed of their eligibility only by their treating physicians. Only participants who have signed all appropriate documentation and who meet participant eligibility requirements will be enrolled. We are completely aware of the difficulties in recruiting participants with this rather rare condition (i.e., HIV-1 related relapsed lymphoma). Therefore, we have proposed an ambitious effort to enroll participants not only from our catchment area of Northern California, but also from other areas of California, as well as from other states. Accrual will be facilitated by a dedicated community advocate, who will initiate and retain interactions with the target community and provide education about the trial. This advocate will interact with clinical research centers as well as other referring clinics across California. He/she will use all available communication tools and specifically the internet to identify and reach out to the target populations for this study through support groups or specific websites, as community outreach materials and methods are specifically approved by the reviewing IRB. All information intended for distribution on the internet or any other media for the purpose of advertising for the study will be approved by the IRB in advance. We have already established a network of collaborators throughout the country and have received verbal and written commitment from some of these centers to propose the study to eligible participants and to facilitate the transfer of their care to our study centers. Entry criteria for cancer studies will be disease, not gender- or ethnicity-specific. Although, participants under 18 and pregnant or nursing women will not be eligible. Information will be disseminated to participants and physicians (e.g., handouts, brochures, etc.), and any advertisement or instruction will be approved by the IRB for the site.
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3.5 participant enrollment procedures

Sites must have this protocol approved by their Institutional Review Boards (IRB) and Institutional BioSafety Committee (IBC) and be registered for study participation with the AMC Operations and Data Management Center (ODMC) before they may enroll participants.
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3.5.1 registration for screening

After an informed consent form has been signed by the participant, the participant must be registered for screening (AMC-097, Segment A) on-line via AdvantageEDCSM. After successful registration into screening, the participant will receive an eleven-digit participant ID and will then enter the screening process (Screening and Pre-entry visits).
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3.5.2 enrollment

After the screening evaluations have been obtained and the participant is determined to be eligible, the participating site will complete the protocol-specific eligibility checklist. **Sites should email the completed (signed by site PI)**
eligibility checklist to mabedi@ucdavis.edu and hsCellularTherapyResearch@ucdavis.edu **for approval by the protocol chair (or**
a designated sub-investigator at UC Davis) before enrolling the participant into AMC-097 Segment B (on-line via AdvantageEDC). Enrollment should occur no more than 1 week prior to, but before stem cell collection (enrollment 1 day prior to or on the day of stem cell collection is strongly encouraged). There should also be a minimum of 2 weeks from the last day of any chemotherapy (except the one used for stem cell mobilization) before a participant can be enrolled on this trial. Once the eligibility checklist is submitted, a system generated confirmation email will be sent to the enroller upon successful completion of the participant enrollment. If the on-line system is inaccessible, the site should notify the AMC ODMC (via email at amcpm@emmes.com or via phone at 301-251-1161) for further instructions. Participants must be enrolled into AMC-097 Segment B prior to receiving HCT.