Sustained analgesic peptide secretion and cell labeling using a novel genetic modification

Shyam Gajavelli, Daniel A. Castellanos, Orion Furmanski, Paul C Schiller, Jacqueline Sagen

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Cell-based therapy for neuropathic pain could provide analgesics to local pain modulatory regions in a sustained, renewable fashion. In order to provide enhanced analgesic efficacy, transplantable cells may be engineered to produce complementary or increased levels of analgesic peptides. In addition, genetic labeling of modified cells is desirable for identification and tracking, but it should be retained intracellularly as desired analgesic peptides are secreted. Usually constructs encode proteins destined for either extra- or intracellular compartments, as these pathways do not cross. However, interactions between intracellular destinations provide a window of opportunity to overcome this limitation. In this report, we have explored this approach using a potential supplementary analgesic peptide, [Ser1]-histogranin (SHG), the stable synthetic derivative of a naturally occurring peptide with N-methyl D-aspartate (NMDA) antagonistic properties. A synthetic SHG gene was combined with (i) nerve growth factor-β (NGF-β) amino-terminal signal peptide to enable secretion, and (ii) a fluorescent cellular label (mRFP) with intervening cathepsin L cleavage site, and subcloned into a lentiviral vector. In addition, an endoplasmic retention signal, KDEL, was added to enable retrieval of mRFP. Using immunocytochemistry and confocal microscopic profile analysis, cells transduced by such lentiviruses were shown to synthesize a single SHG-mRFP polypeptide that was processed, targeted to expected subcellular destinations in several cell types. Dot blot and Western analysis revealed stable transduction and long-term secretion of SHG from PC12 cells in vitro. Transplantation of such cells provided modest analgesia in a rodent pain model consistent with low levels of SHG peptide in the cerebrospinal fluid (CSF). These results suggest that it is possible to deliver proteins with different final destinations from a single construct, such as pharmacologically active peptide for secretion and intracellular label for identifying transplantable cells.

Original languageEnglish
Pages (from-to)445-455
Number of pages11
JournalCell Transplantation
Volume17
Issue number4
StatePublished - May 28 2008

Fingerprint

Labeling
Peptides
Analgesics
Labels
Cerebrospinal fluid
Proteins
Cathepsin L
Polypeptides
Pain
Lentivirus
PC12 Cells
Cell Transplantation
Neuralgia
Nerve Growth Factor
N-Methylaspartate
Cell- and Tissue-Based Therapy
Protein Sorting Signals
Analgesia
Genes
Cerebrospinal Fluid

Keywords

  • Histogranin
  • Lentiviral
  • NMDA antagonist
  • Pain
  • PC12 cells
  • Transplantation

ASJC Scopus subject areas

  • Cell Biology
  • Transplantation

Cite this

Sustained analgesic peptide secretion and cell labeling using a novel genetic modification. / Gajavelli, Shyam; Castellanos, Daniel A.; Furmanski, Orion; Schiller, Paul C; Sagen, Jacqueline.

In: Cell Transplantation, Vol. 17, No. 4, 28.05.2008, p. 445-455.

Research output: Contribution to journalArticle

Gajavelli, Shyam ; Castellanos, Daniel A. ; Furmanski, Orion ; Schiller, Paul C ; Sagen, Jacqueline. / Sustained analgesic peptide secretion and cell labeling using a novel genetic modification. In: Cell Transplantation. 2008 ; Vol. 17, No. 4. pp. 445-455.
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