Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs

Abstract

Here we describe an entirely new class of cell-penetrating peptide (CPP) represented by the short peptide Xentry (LCLRPVG) derived from an N-terminal region of the X-protein of the hepatitis B virus. Xentry permeates adherent cells using syndecan-4 as a portal for entry, and is uniquely restricted from entering syndecan-deficient, non-adherent cells, such as resting blood cells. Intravenous injection of Xentry alone or conjugated to β-galactosidase led to its delivery to most tissues in mice, except circulating blood cells. There was a predilection for uptake by epithelia. Anti-B-raf antibodies and siRNAs linked to Xentry were capable of killing B-raf-dependent melanoma cells. Xentry represents a new class of CPP with properties that are potentially advantageous for life science and therapeutic applications.

Direct Cytosolic Delivery of Polar Cargo to Cells by Spontaneous Membrane-translocating Peptides

“…After cooling, samples were centrifuged at 14,000 rpm in an Eppendorf centrifuge to pellet undigested debris. Similar extraction procedures have been reported by other groups (37)(38)(39)(40). The supernatants were added to the wells of a 96-well plate at dilutions of 1, 1:2, 1:4, 1:8, and 1:16.…”

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

“…11 Although cargo proteins that are internalized by clathrinmediated endocytosis often route to late endosomes and subsequently undergo lysosomal degradation (or routes to recycling endosomes), 7,11 this is not always the case, as exemplified by cytosolic release of some CPPs or toxins after cellular uptake by clathrin-mediated endocytosis. 9,37,44,45 Several lines of evidence, such as intracellular distribution and trafficking studies by confocal microscopy, direct access of KT4 to cytosolic KRS proteins, and calcein release assay, indicate that endocytosed cytotransmab is directly released to cytosol from early endosomes, passing over lysosome, ER, Golgi, and nucleus trafficking. However, the underlying molecular mechanism by which cytotransmab escapes from early endosomes into the cytosol is not clear.…”

“…1,2,7 Conjugation with protein transduction domains, which are represented by cell-penetrating peptides (CPPs) such as the HIV-1 TAT peptide, has been extensively attempted in order to facilitate the intracellular delivery of antibodies formatted as single chain variable fragments (scFvs), antigen-binding fragments (Fabs), and full-length IgGs. [8][9][10] However, most of the CPP-conjugated antibodies inherited the intrinsic intracellular trafficking of the parent CPPs, which were either entrapped inside endocytic vesicles, translocated into the nucleus, or eventually degraded in lysosomes without efficient endosomal release into the cytosol. 1,10,11 Some anti-DNA polyclonal antibodies or monoclonal antibodies (mAbs) predominantly found in humans and mice with autoimmune diseases have been shown to possess the ability to penetrate into living cells in their IgG format.…”

Viral and Other Cell-Penetrating Peptides as Vectors of Therapeutic Agents in Medicine

“…Peptide/protein and nucleic acids were delivered into the cells, both in vitro and in vivo, respectively (Morris et al, 2008). Since that time, the new CPP-based carrier-cargo complexes have been under intensive development (Jafari et al, 2013;Montrose et al, 2013;Puig-Saus et al, 2014).…”

“…The noncovalent strategy is a suitable method for delivering negatively charged ONs such as siRNA (Crowet et al, 2013;Jafari et al, 2013), plasmid DNA (Liu et al, 2013b), antisense ONs, and splicing correction ONs (Margus et al, 2012), as well as peptides and proteins (Myrberg et al, 2007;Montrose et al, 2013). However, for the latter, the noncovalent strategy for cargo and carrier has been used less often than the covalent one.…”