Intracellular trafficking of a dynein-based nanoparticle designed for gene delivery.

The success of viruses in the delivery of the viral genome to target cells relies on the evolutionary selection of protein-based domains able to hijack the intermolecular interactions through which cells respond to intra- and extracellular stimuli. In an effort to mimic viral infection capabilities during non-viral gene delivery, a modular recombinant protein named T-Rp3 was recently developed, containing a DNA binding domain, a dynein molecular motor interacting domain, and a TAT-derived transduction domain. Here, we analyzed at the microscopic level the mechanisms behind the cell internalization and intracellular trafficking of this highly efficient modular protein vector. We found that the protein has the ability to self-assemble in discrete protein nanoparticles resembling viral capsids, to bind and condense plasmid DNA (pDNA), and to interact with eukaryotic cell membranes. Confocal and single particle tracking assays performed on living HeLa cells revealed that the T-Rp3 nanoparticles promoted an impressive speed of cellular uptake and perinuclear accumulation. Finally, the protein demonstrated to be a versatile vector, delivering siRNA at efficiencies comparable to Lipofectamine™. These results demonstrate the high potential of recombinant modular proteins with merging biological functions to fulfill several requirements needed to obtain cost-effective non-viral vectors for gene-based therapies.

A cytoplasm-sensitive peptide vector cross-linked with dynein light chain association sequence (DLCAS) enhances gene expression.

We previously engineered a novel, non-viral, multifunctional gene vector (STR-CH(2)R(4)H(2)C) containing stearoyl (STR) and a block peptide consisting of Cys (C), His (H), and Arg (R). STR-CH(2)R(4)H(2)C forms a nano-complex with pDNA and is stabilized by electronic interactions and disulfide cross linkages. In blood, pDNA, a cytosol-sensitive gene vector, is released from the complex into the cytosol. The current study aimed to make STR-CH(2)R(4)H(2)C capable of active nuclear localization. The dynein light chain association sequence (DLCAS) was disulfide cross-linked to STR-CH(2)R(4)H(2)C/pDNA through disulfide linkages, and the gene expression ability of this DLCAS cross-linked gene vector was evaluated. We examined the gene transfection efficiency of S-180 cells transfected with the STR-CH(2)R(4)H(2)C/DLCAS/pDNA complex. STR-CH(2)R(4)H(2)C/DLCAS/pDNA showed significantly higher and faster gene expression compared with STR-CH(2)R(4)H(2)C/pDNA. We also evaluated the cellular uptake ability of STR-CH(2)R(4)H(2)C/DLCAS/Cy5-labeled pDNA complex. STR-CH(2)R(4)H(2)C/DLCAS/pDNA showed significantly lower cellular uptake compared with STR-CH(2)R(4)H(2)C/pDNA. This result indicates that high gene expression of STR-CH(2)R(4)H(2)C/DLCAS/pDNA does not facilitate its cellular uptake. In addition, the gene expression of DLCAS/STR-CH(2)R(4)H(2)C/pDNA in S-180 cells pretreated with the tubulin polymerization inhibitor, nocodazole (NCZ), was significantly lower than that in the absence of NCZ. These results indicate that the high transfection efficiency of DLCAS/STR-CH(2)R(4)H(2)C/pDNA is dependent on intra-cellular transport utilizing the microtubule motor protein, dynein. Taken together, our results suggest that DLCAS-modified STR-CH(2)R(4)H(2)C may be a promising gene delivery system.

Ciliary beating recovery in deficient human airway epithelial cells after lentivirus ex vivo gene therapy.

Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1-deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT-PCR and western blot, respectively. Human airway epithelial cells that were DNAI1-deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease.

Cytoplasmic dynein and dynactin are required for the transport of microtubules into the axon.

Previous work from our laboratory suggested that microtubules are released from the neuronal centrosome and then transported into the axon (Ahmad, F.J., and P.W. Baas. 1995. J. Cell Sci. 108: 2761-2769). In these studies, cultured sympathetic neurons were treated with nocodazole to depolymerize most of their microtubule polymer, rinsed free of the drug for a few minutes to permit a burst of microtubule assembly from the centrosome, and then exposed to nanomolar levels of vinblastine to suppress further microtubule assembly from occurring. Over time, the microtubules appeared first near the centrosome, then dispersed throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. In the present study, we microinjected fluorescent tubulin into the neurons at the time of the vinblastine treatment. Fluorescent tubulin was not detected in the microtubules over the time frame of the experiment, confirming that the redistribution of microtubules observed with the experimental regime reflects microtubule transport rather than microtubule assembly. To determine whether cytoplasmic dynein is the motor protein that drives this transport, we experimentally increased the levels of the dynamitin subunit of dynactin within the neurons. Dynactin, a complex of proteins that mediates the interaction of cytoplasmic dynein and its cargo, dissociates under these conditions, resulting in a cessation of all functions of the motor tested to date (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132: 617-633). In the presence of excess dynamitin, the microtubules did not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, we conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon.