The Sangamo Domain: Cross-Clade Inhibition of HIV on Primary Cells by CXCR4 or or CCR5 Fused to the C34 Peptide from gp41 HR2

Wang1, GJ Leslie2, J DeClercq1, MW Richardson2, APO Jordon2, PD Gregory1, JL

Riley2, JA Hoxie2, MC Holmes1

1Sangamo Biosciences Inc. Richmond, CA, USA; 2University of Pennsylvania, Philadelphia, PA, USA

HIV-1 entry into CD4+ T cells requires binding to CD4 and either the CCR5 (R5)

or CXCR4 (X4) co-receptor. Thus, strategies that disable productive co-receptor

(CoR) engagement should provide potent protection from HIV infection.

Previously we described a 34 amino acid peptide from the C-terminal heptad

repeat-2 domain of gp41 (C34) which, when fused to the amino terminus (NT)

of either R5 or X4, inhibits HIV-1 infection in transformed cells in vitro. Moreover,

our initial studies suggested that C34-R5 or C34-X4 fusions provided transdominant

resistance to infection irrespective of viral tropism (i.e. either C34-R5

or C34-X4 could inhibit entry of R5, X4 or dual-tropic isolates).

Here we demonstrate that C34-R5 or C34-X4 expression by lentiviral

transduction in primary CD4 T-cells from multiple donors results in almost

complete inhibition (>98%) of HIV-1 infection based on intracellular p24 levels

and RT activities. GFP-only and C34-CD4 expressing cells were infected at

levels similar to untransduced T-cells. C34/CoR expression was >90% on Day

0 and stable during the 14 days of culture (>85%). Trans-dominant inhibition by

C34-R5 or C34-X4 occurred for X4, R5 and dual-tropic primary isolates from

clades B and A/E. Remarkably, when C34-CoR transduced and untransduced

cells were mixed (1:4, respectively) and challenged with diverse HIV isolates, a

condition that provides a more sustained exposure to HIV, selective enrichment

of C34-CoR expressing cells occurred from the expected starting levels of ~25%

up to 60% C34-CoR+ cells during viral replication. Lastly, PMA/ionomycin and

anti-CD3/CD28 stimulation of C34-R5 and C34-X4 expressing T-cells resulted

in the expected robust expansion of these T cells which produced levels of

intracellular IFNg, MIP-1b, TNFa, and IL-2 that were indistinguishable from

untransduced cells. This novel method of engineering HIV-resistant, functional

CD4 T-cells that can be expanded ex vivo and adoptively reinfused represents a

promising and innovative approach with the potential to control HIV infection in

humans.

The Sangamo Domain