Relative Mitochondrial Toxicity of Tenofovir Alafenamide (TAF) vs. Tenofovir Disoproxil Fumarate (TDF)

Increased comorbidities such as cardiovascular disease (CVD), bone and kidney disease are
emerging problems in HIV infection but the mechanisms are unclear. Understanding how
antiretrovirals can minimize morbidity in treated HIV infection is a research priority.
Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) are included in all HIV
treatment regimens. Tenofovir (TFV) disoproxil fumarate (TDF) has been associated with an
increased risk of nephrotoxicity and bone disease compared with other NRTIs. Tenofovir
alafenamide (TAF) is an oral prodrug of TFV, but is much more stable in plasma as compared
with TDF and markedly lower plasma levels of TFV are thought to lead to the favorable safety
profile of TAF. Mitochondrial dysfunction has a key role in HIV pathogenesis and may be the
common denominator that drives pathogenesis of several comorbidities. Despite the better
safety profile of newer (such as TDF) compared to older NRTIs, there remain concerns for the
potential for longer term toxicity of the current NRTIs especially since the exact cellular
effects of NRTIs remain unclear. It is unknown whether a four-fold increase in intracellular
drug levels seen in peripheral blood mononuclear cells (PBMCs) with TAF may increase toxicity
in mitochondria. Better understanding of these effects could provide insights into mechanisms
of HIV pathogenesis and selection of NRTIs that improve morbidity in chronic HIV infection.

Hypothesis: Despite higher intracellular levels, TAF has minimal/no overall mitochondrial
toxicity compared to TDF in vivo. The investigators anticipate that the preliminary data and
the proposed experimental approach will set the basis for future large scale studies to
directly compare subtle potential mitochondrial toxicities of newer NRTIs in large HIV
cohorts.

Primary Objective: This research application will explore the relative mitochondrial toxicity
of newer NRTIs (TAF, TDF) as a possible mechanism for differential NTRI-related toxicities.
These data will allow selection of NRTIs that may improve morbidity in chronic treated HIV
infection, given the known possible role of mitochondrial dysfunction in pathogenesis of
HIV-related comorbidities. Towards this aim, the investigators will use a robust experimental
approach to study NRTI-related mitochondrial dysfunction using novel methods, human cell
lines and PBMC. The specific aims of this project are: Aim 1: To evaluate the relative in
vitro effects of TAF and TDF compared to an older NRTI (ddC) on 5 independent measures of
mitochondrial function in the human cell line HepG2 and PBMC. Hypothesis: Despite higher
intracellular levels, TAF has minimal/no overall mitochondrial toxicity compared to TDF in
both the HepG2 line and PBMC. ddC will be used as a positive control for NRTI-induced
mitochondrial dysfunction. Aim 2: To explore in vivo whether there is increased mitochondrial
dysfunction with the use of TAF vs. TDF in chronic treated HIV infection. The overall goal of
this proposal is to determine the in vitro and in vivo effects of TAF on mitochondria.

Treatment regimen: Healthy HIV-uninfected > 18 years old with no known comorbidities
including metabolic disease will be recruited for PBMC isolation for the in vitro studies
described in Aim 1 (n=30). For Aim 1, cells will be exposed to 10 μM (and other dose that
will be determined after discussions with Gilead) NRTIs (TDF, TAF, ddC) for up to 14 days of
treatment. To evaluate the relative mitochondrial toxicity of TDF vs. TAF in vivo (Aim 2), an
open-label switch study will be conducted in virologically-suppressed, HIV-1 positive
participants. The switch regimen will be Stribild® (elvitegravir 150 mg/cobicistat 150
mg/emtricitabine 200 mg/tenofovir disoproxil fumarate 300 mg; E/C/F/TDF) to Genvoya®
(elvitegravir 150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir alafenamide 10 mg;
E/C/F/TAF). For inclusion and exclusion criteria see respective sections.

Primary Endpoint: Overall, the above in vitro and in vivo measurements of mitochondrial
function before and after TAF and TDF will test the hypotheses that newer NRTIs have minimal
overall mitochondrial toxicity in humans. From all the measures of mitochondrial function,
cellular oxygen consumption (COC; as determined by Seahorse technology) will be considered
the "gold standard" given it is a measure of the most important mitochondrial function
(oxidative phosphorylation) that relates to clinical outcome. Given the uncertainty of what
measure of mitochondrial function can best predict NRTI-induced mitochondrial toxicity,
comparison of relative fold induction of mitochondrial dysfunction in PBMC vs. HepG2 cells in
response to a specific concentration of a known NRTI inducer of mitochondrial dysfunction
(ddC) will be used to guide analysis. The baseline measurement will be used to control for
confounders of NRTI-related mitochondrial dysfunction (each person will serve as his/her own
control) and results will be expressed as relative-fold change compared to baseline. In
addition, using similar approach, Aim 2 will explore whether switch from TDF to a TAF-based
regimen will induce any changes in measures of mitochondrial function. This approach ensures
that any changes in the mitochondrial function at 6 and/or 12 months will be secondary only
to the NRTI switch.

Secondary Endpoints: It is accepted that two independent measures of mitochondrial function
can more reliably predict mitochondrial dysfunction compared to each one of the measures
separately. The investigators will define NRTI-induced mitochondrial toxicity as significant
change in any of other major measures of mitochondrial toxicity [reduction in membrane
potential (Δψm), increase in mitochondrial reactive oxygen species (mito-ROS), decrease in
the amount of mitochondrial DNA (mtDNA), abnormal mitochondrial dynamics (increase
fragmentation of mitochondria)). Finally, other exploratory secondary endpoints (based on
available left-over resources) will be measurement of intracellular levels of TAF
metabolites. Flow cytometry and live cell imaging will be determined as needed.

The investigators will define NRTI-induced mitochondrial toxicity as significant change in
any of the measures of mitochondrial toxicity (COC, Δψm, mito-ROS, mtDNA, mitochondrial
dynamics). The baseline measurement will be used to control for confounders of NRTI-related
mitochondrial dysfunction (each person will serve as his/her own control) and results will be
expressed as relative-fold change compared to baseline. In addition, using similar approach,
Aim 2 will explore whether switch from TDF to a TAF-based regimen will induce any changes in
measures of mitochondrial function. This approach ensures that any changes in the
mitochondrial function at 6 and/or 12 months will be secondary only to the NRTI switch.

Since each donor will contribute samples to all experimental groups (baseline, follow up
measurement), paired t-test, linear mixed effects model and nonparametric test (as needed)
will be used to compare expression measures between groups adjusting for donor effects (age,
sex, race.

The results from this research will yield useful information on NRTI-related overall
mitochondrial dysfunction that can further be validated in large studies with data on
clinical outcomes.

Inclusion Criteria:

- Inclusion criteria for HIV negative participants

All HIV negative participants must meet the following criteria:

1. 18 years of age or older

2. Understands and agrees to local STI reporting requirements

3. HIV negative at screening by self-report, with no significant medical diagnoses

4. HIV negative by both HIV antibody and HIV PCR in blood (samples obtained during Visit
1)

5. Able and willing to communicate in English

6. Able and willing to provide written informed consent to take part in the study

7. Able and willing to provide adequate information for locator purposes

8. Able and willing to provide medical/surgical history

9. Availability to return for all study visits, barring unforeseen circumstances

10. Willing to abstain from insertion of anything (drug, enema, penis or sex toy) in
rectum for 12 hours before and 72 hours after each flexible sigmoidoscopy

In addition to the criteria listed above, female participants must meet the following
criteria

11. Negative pregnancy test

12. Post-menopausal or using and acceptable form of contraception (e.g. barrier method,
IUD, hormonal contraception, or surgical sterilization).

HIV participant inclusion criteria

Specific enrollment inclusion criteria include:

- 18 years of age or older

- Cases: Chronically infected and on anti-retroviral therapy with suppressed viremia
(viral RNA <50 copies per ml)

- On stable antiretroviral therapy for 6 months with either a) Stribild®; elvitegravir
150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir disoproxil fumarate 300 mg;
E/C/F/TDF)

- Adequate renal function determined by the Cockcroft-Gault formula for creatinine
clearance (>60 mL/min/1.73 m2

- Controls: HIV negative as documented in prior tests and willing to undergo repeat HIV
testing

- Able and willing to communicate in English

- Able and willing to provide written informed consent to take part in the study

Exclusion Criteria:

- Exclusion criteria for all study participants:

Participants who meet any of the following criteria at screening will be excluded from the
study:

1. History of chronic inflammatory bowel disease, radiation proctitis or other chronic
gastrointestinal disease, exclusive of functional bowel disease (irritable bowel
syndrome)

2. History of significant gastrointestinal bleeding

3. History of a bleeding disorder

4. History of colostomy

5. History of auto-immune diseases

6. Chronic viral hepatitis

7. History of diabetes

8. History of chemotherapy (for cancer or organ transplantation)

9. The chronic or recent (~2 weeks) use of antimitotic drugs, sulfonamides or antibiotics

10. History of or current coagulopathy and/or on anticoagulant therapy

11. Anticipated use or unwillingness to abstain from use of aspirin, NSAIDS or any other
drugs (including over the counter products) that are associated with the increased
likelihood of bleeding

12. Active rectal infection (gonorrhea, chlamydia trachomatis, or HSV)

13. Positive STI at screening (urine NAAT)

o Participants will be allowed one re-screening visit after appropriate STI treatment

14. History of underlying cardiac arrhythmia or renal disease

15. History of severe recent cardiac or pulmonary event

16. > Grade 2 laboratory abnormality at baseline that can not be documented as stable
/chronic for that individual

17. Any other condition or prior therapy that, in the opinion of the investigator, would
make the patient unsuitable for the study or unable to comply with study requirements.
Such conditions may include, but are not limited to, current or recent history of
severe, progressive, or uncontrolled renal, hepatic, hematological, gastrointestinal,
endocrine, pulmonary, neurological or cerebral disease.

18. Pregnancy

19. Breastfeeding

20. Female of child-bearing potential unwilling to use acceptable form of contraception