HIV Sterilizing Cure: Full-Stack Proof-of-Concept Protocol

Construct Synthesis

• dCas9-KRAB-MeCP2 (plasmid + mRNA)
• sgRNA library: 20-30 guides targeting HIV LTR
  • NF-κB binding sites
  • Sp1 binding sites
  • TAR region
• TAR-reporter constructs (pSentinel-1, -2, -3)
• LTR-iCasp9 kill switch

1. J-Lat Reactivation

• Seed 2×10⁵ cells/well
• TNF-α (10 ng/mL) or PMA (10 nM), 24h
• Flow: Live → Singlets → GFP+
• QC: Vehicle <5% GFP+, TNF-α >30% GFP+

2. Primary CD4+ Isolation

• Ficoll gradient → negative selection
• QC: >95% CD4+ CD3+, >95% viability

3. p24 ELISA

• Standard curve R² >0.99
• CV <15%

4. ChIP-qPCR

• Sonicate to 200-500 bp
• H3K9me3 enrichment >5× over IgG

Protocol 1.1: J-Lat sgRNA Screen (Months 4-5)

Objective: Identify top sgRNAs for LTR silencing

Controls:

• Untransfected
• Non-targeting sgRNA
• dCas9 only (no KRAB)

Success Criteria:

• ≥90% reduction in GFP+ vs control
• ≥80% silencing maintained at day 28
• H3K9me3 enrichment >5× at LTR

Output: Top 3-5 sgRNAs for advancement

Protocol 1.2: Primary CD4+ Validation (Months 6-7)

Objective: Confirm silencing in primary cells

Success Criteria:

• >80% p24 reduction vs untreated
• Integrated DNA unchanged (no killing)
• Viability >90%

Donors: n=3 minimum, independent experiments

Protocol 1.3: Multi-Strain Test (Month 7)

Strains: NL4-3, BaL, 92UG037, 93IN101

Procedure: Repeat Protocol 1.1 with each strain using lead sgRNA panel

Success Criteria: >70% silencing across all clades

Protocol 2.1: Tat-Responsive Reporter (Months 6-8)

Constructs:

• pSentinel-1: TAR → minimal promoter → GFP
• pSentinel-2: TAR → minimal promoter → secreted NanoLuc
• pSentinel-3: TAR → minimal promoter → surface CD marker

Week 1-2: HEK293T Characterization

1. Transfect pSentinel + Tat plasmid (dose range)
2. Measure reporter at 24h, 48h
3. Calculate signal/noise ratio
4. Target: S/N >100:1

Week 3-4: J-Lat Integration

1. Package pSentinel as lentivirus
2. Transduce J-Lat, select stable lines
3. TNF-α challenge
4. Correlate sentinel vs HIV-GFP

Success Criteria:

• S/N >50:1
• r² >0.9 correlation with HIV reactivation
• False positive rate <1%

Protocol 2.2: Kill Switch Validation (Month 8-9)

Construct: LTR → iCasp9

Procedure:

1. Transfect HEK293T: LTR-iCasp9 + Tat → measure apoptosis (Annexin V)
2. Integrate into J-Lat → TNF-α → selective killing of GFP+ cells
3. Primary cells: confirm bystander killing <5%

Safety Check: No cytokine release (IL-6, TNF-α, IFN-γ)

Protocol 3.1: Cell Panel Establishment (Months 8-9)

QC: Purity >95%, infectability confirmed, bank for 12+ experiments

Protocol 3.2: Latency Establishment (Month 9-10)

CD4+ T Cells:

• Activate → infect MOI 0.1 → rest in IL-7
• Confirm: p24 low, integrated DNA+, reactivatable

Macrophages:

• Infect with BaL (MOI 0.5) → 7d productive → wash → rest
• Confirm: minimal p24, integrated DNA+

Microglia:

• VSV-G pseudotyped virus, low MOI
• Maintain resting conditions

Protocol 3.3: Cross-Reservoir Validation (Month 10-11)

Matrix Experiment:

Apply Layer 1 + Layer 2 to each reservoir type and test efficacy across CD4+ T cells, macrophages, and microglia.

Detection Methods (apply all, select best):

• Tat activity reporter
• Spliced/unspliced RNA ratio (ddPCR)
• p24 flow cytometry
• Surface Env (bNAb staining)

Protocol 4.1: Standard Selective Pressure (Months 9-10)

Setup:

• MT-2 cells + NL4-3 (MOI 0.01)
• Allow infection to establish (p24 >100 ng/mL)
• Apply Layer 1 at 50% dose
• Passage every 3-4 days × 20 passages
• n=5 independent lineages

At Each Passage:

• Monitor p24
• If breakthrough: harvest, sequence LTR/Tat/Rev

After 20 Passages:

• Deep sequence entire proviral genome (>10,000× coverage)
• Identify enriched mutations
• Test candidates against full-dose intervention

Controls:

• No intervention (baseline evolution)
• EFV drug control (known escape kinetics)

Protocol 4.2: Hypermutation Stress Test (Month 10-11)

Maximally stress viral evolution:

1. APOBEC3G-overexpressing cells + Vif-deficient HIV
2. Allow extensive G→A hypermutation
3. Apply Layer 1+2
4. Deep sequence survivors

Expected: Hypermutation destroys function faster than generating escape

Protocol 4.3: Diverse Strain Panel (Month 11)

Repeat Protocol 4.1 with: NL4-3, BaL, 92UG037, 93IN101

Expected: Zero escape across all clades

Protocol 4.4: Long-Term Escape Watch (Months 10-12, ongoing)

• Primary CD4+ T cells, latently infected
• Full Layer 1 + Layer 2
• Maintain 6-12 months
• Monthly reactivation challenge
• Sequence any breakthrough

Protocol 5.1: Cytotoxicity Panel (Month 10)

Cell Types: Primary CD4+ T, PBMC, HepG2, iPSC-cardiomyocytes

Doses: 0.1×, 1×, 10×, 100× therapeutic

Acceptance:

• Viability >95% at 1× dose
• Viability >80% at 10× dose
• No apoptosis increase at 1× dose

Protocol 5.2: Genotoxicity Screen (Month 11)

Assays:

1. γH2AX Foci
   • Treat → fix at 24h, 48h → immunostain → count foci
   • Compare to etoposide positive control
   • Accept: No elevation vs mock
2. Comet Assay
   • Treat 24h → embed → electrophorese → quantify tail DNA
   • Accept: <15% tail DNA
3. Karyotyping
   • Treat 72h → metaphase arrest → analyze spreads
   • Accept: No aberrations

Protocol 5.3: Immunotoxicity Panel (Month 12)

Cytokine Release:

• Treat PBMC → supernatant at 4h, 24h, 48h
• Luminex panel: IFN-γ, TNF-α, IL-1β, IL-6, IL-2, IL-10, IL-4
• Accept: No elevation >2× baseline

T Cell Function:

• Pre-treat → stimulate anti-CD3/CD28 → measure proliferation, IFN-γ
• Accept: >80% of control

NK Function:

• Pre-treat → K562 co-culture → measure killing
• Accept: >80% of control

Protocol 5.4: Off-Target Silencing (Month 12)

RNA-seq:

• Treat primary CD4+ (HIV-negative) at 1× dose
• Harvest at 48h, 7d, 14d
• Sequence 30M reads/sample
• DESeq2: |log2FC| >1, padj <0.05

Accept: <10 differentially expressed genes, none in critical pathways

Test:

1. Layer 1 only → measure 60-day durability
2. Layer 1 + Layer 2 → measure 60-day durability
3. Compare breakthrough rates

Validation Assay:

• Long-term culture with periodic reactivation challenge
• Flow: surveillance reporter + HIV markers
• Cell counts (no unexpected depletion)

Pass Criteria: Combined durability > Layer 1 alone

Test:

1. Generate putative escape variants (from Layer 4)
2. Spike into latent culture
3. Monitor by Layer 2 surveillance
4. Deep sequence

Pass Criteria: Any Layer 1 escapee caught by Layer 2/3

Procedure:

1. PBMC from 5-10 ART-suppressed patients
2. Isolate CD4+ T cells
3. Apply integrated system
4. Compare reservoir quantification: standard (QVOA) vs invariant detection
5. Reactivate subset, track by both methods

Efficacy Study (Month 16-17)

Model: BLT or NSG-hu mice, HIV-infected, ART-suppressed

Design:

• Groups: Vehicle (n=8), Treatment (n=8)
• Administer integrated intervention
• Withdraw ART
• Monitor viral rebound for 60+ days

Endpoints:

• Plasma viral load (weekly)
• Cell-associated HIV DNA/RNA
• Human immune cell counts

Success: No rebound in treatment group for >60 days post-ART

Safety Study (Month 17-18)

Design:

• Groups: Vehicle, 1×, 3×, 10× dose (n=8-10 each)
• 28-day observation

Endpoints:

• Body weight (daily)
• Blood chemistry (day 0, 7, 14, 28)
• Hematology
• Human immune cells (flow)
• Necropsy + histopathology

Accept:

• No mortality
• Weight loss <10%
• Blood chemistry normal
• No histopathology findings

Go Criteria:

• Efficacy in humanized mice
• Tylenol-level safety confirmed
• Zero escape under all tested conditions
• Clear regulatory path
• Sufficient IP position
• Financing path identified

No-Go Criteria:

• Insufficient efficacy
• Unresolvable safety signals
• Escape variants emerge
• Regulatory path blocked
• IP position untenable