Gene therapy to inhibit xenoantibody production using lentiviral vectors in non-human primates.

Xenoantibodies to the gal alpha1,3 gal (gal) epitope impede the use of pig tissues for xenotransplantation, a procedure that may help overcome the shortage of human organ donors. Stable gal chimerism and tolerance to gal(+) hearts could be achieved in alpha1,3-galactosyltransferase (alpha1,3GT)(-/-) mice using lentiviral vectors expressing porcine alpha1,3GT, the enzyme that synthesizes the gal carbohydrate. In this study, we evaluated whether chimerism sufficient to inhibit anti-gal xenoantibody responses can be achieved using lentivectors in non-human primates. Rhesus macaques were transplanted with autologous, alpha1,3GT-transduced bone marrow (BM) following sublethal irradation. Simian immunodeficiency virus (SIV)- and human immunodeficiency virus (HIV)-1-derived lentiviral constructs were compared. Chimerism was observed in several hematopoietic lineages in all monkeys. Engraftment in animals receiving SIV-based alpha1,3GT constructs was similar to that achieved using the HIV-1-derived lentivector for the first 2 months post-transplantation, but increased thereafter to reach higher levels by 5 months. Upon immunization with porcine hepatocytes, the production of anti-gal immunoglobulin M xenoantibody was substantially reduced in the gal(+) BM recipients compared to controls. This study is the first to report the application of gene therapy to achieve low-level, long-term gal chimerism sufficient to inhibit production of anti-gal antibodies after immunization with porcine cells in rhesus macaques.

Expression changes in tolerant murine cardiac allografts after gene therapy with a lentiviral vector expressing alpha1,3 galactosyltransferase.

Comparison of intragraft gene expression changes in tolerant cardiac allograft models may provide the basis for identifying pathways involved in graft survival. Our laboratory has previously demonstrated that tolerance to the gal alpha1,3 gal epitope, the major target of rejection of wild-type pig hearts in human cardiac transplantation, can be achieved after transplantation with bone marrow transduced with a lentiviral vector expressing alpha1,3 galactosyltransferase. We now present intracardiac gene expression changes associated with long-term tolerance in this model. Biotin-labeled cRNA was hybridized to Affymetrix GeneChip 430 2.0 Mouse Genome Arrays. Data were subjected to functional annotation analysis to identify genes of known function in which expression was increased or decreased by at least 2-fold (t-test, P < .05) in tolerant gal+/+ wild-type hearts as compared to transplanted syngeneic controls. Tolerant hearts demonstrated increased expression of genes associated with the stress response, modulation of immune function and cell survival (HSPa9a, CD56, and Akt1s1), and decreased expression of several immunoregulatory genes (CD209, CD26, and PDE4b). These data suggest that tolerance may be associated with activation of immunomodulatory and survival pathways.

Human myoblast fusion requires expression of functional inward rectifier Kir2.1 channels.

Myoblast fusion is essential to skeletal muscle development and repair. We have demonstrated previously that human myoblasts hyperpolarize, before fusion, through the sequential expression of two K+ channels: an ether-à-go-go and an inward rectifier. This hyperpolarization is a prerequisite for fusion, as it sets the resting membrane potential in a range at which Ca2+ can enter myoblasts and thereby trigger fusion via a window current through alpha1H T channels.

T-type alpha 1H Ca2+ channels are involved in Ca2+ signaling during terminal differentiation (fusion) of human myoblasts.

Mechanisms underlying Ca(2+) signaling during human myoblast terminal differentiation were studied using cell cultures. We found that T-type Ca(2+) channels (T-channels) are expressed in myoblasts just before fusion. Their inhibition by amiloride or Ni(2+) suppresses fusion and prevents an intracellular Ca(2+) concentration increase normally observed at the onset of fusion. The use of antisense oligonucleotides indicates that the functional T-channels are formed by alpha1H subunits. At hyperpolarized potentials, these channels allow a window current sufficient to increase [Ca(2+)](i). As hyperpolarization is a prerequisite to myoblast fusion, we conclude that the Ca(2+) signal required for fusion is produced when the resting potential enters the T-channel window. A similar mechanism could operate in other cell types of which differentiation implicates membrane hyperpolarization.

Mibefradil (Ro 40-5967) inhibits several Ca2+ and K+ currents in human fusion-competent myoblasts.

1. The effect of mibefradil (Ro 40-5967), an inhibitor of T-type Ca2+ current (I(Ca)(T)), on myoblast fusion and on several voltage-gated currents expressed by fusion-competent myoblasts was examined. 2. At a concentration of 5 microM, mibefradil decreases myoblast fusion by 57%. At this concentration, the peak amplitudes of I(Ca)(T) and L-type Ca2+ current (I(Ca)(L)) measured in fusion-competent myoblasts are reduced by 95 and 80%, respectively. The IC50 of mibefradil for I(Ca)(T) and I(Ca)(L) are 0.7 and 2 microM, respectively. 3. At low concentrations, mibefradil increased the amplitude of I(Ca)(L) with respect to control. 4. Mibefradil blocked three voltage-gated K+ currents expressed by human fusion-competent myoblasts: a delayed rectifier K+ current, an ether-à-go-go K+ current, and an inward rectifier K+ current, with a respective IC50 of 0.3, 0.7 and 5.6 microM. 5. It is concluded that mibefradil can interfere with myoblast fusion, a mechanism fundamental to muscle growth and repair, and that the interpretation of the effect of mibefradil in a given system should take into account the action of this drug on ionic currents other than Ca2+ currents.

An ether -à-go-go K+ current, Ih-eag, contributes to the hyperpolarization of human fusion-competent myoblasts.

1. Two early signs of human myoblast commitment to fusion are membrane potential hyperpolarization and concomitant expression of a non-inactivating delayed rectifier K+ current, IK(NI). This current closely resembles the outward K+ current elicited by rat ether-à-go-go (r-eag) channels in its range of potential for activation and unitary conductance. 2. It is shown that activation kinetics of IK(NI), like those of r-eag, depend on holding potential and on [Mg2+]o, and that IK(NI), like r-eag, is reversibly inhibited by a rise in [Ca2+]i. 3. Forced expression of an isolated human ether-à-go-go K+ channel (h-eag) cDNA in undifferentiated myoblasts generates single-channel and whole-cell currents with remarkable similarity to IK(NI). 4. h-eag current (Ih-eag) is reversibly inhibited by a rise in [Ca2+]i, and the activation kinetics depend on holding potential and [Mg2+]o. 5. Forced expression of h-eag hyperpolarizes undifferentiated myoblasts from -9 to -50 mV, the threshold for the activation of both Ih-eag and IK(NI). Similarly, the higher the density of IK(NI), the more hyperpolarized the resting potential of fusion-competent myoblasts. 6. It is concluded that h-eag constitutes the channel underlying IK(NI) and that it contributes to the hyperpolarization of fusion-competent myoblasts. To our knowledge, this is the first demonstration of a physiological role for a mammalian eag K+ channel.

Cloning of a human ether-a-go-go potassium channel expressed in myoblasts at the onset of fusion.

An early sign of human myoblast commitment to fusion is the expression of a non-inactivating delayed rectifier K+ current, I(K(NI)), and an associated membrane potential hyperpolarization. We have isolated the full-length coding region of a human ether-a-go-go K+ channel (h-eag) from myoblasts undergoing differentiation. The h-eag gene was localized to chromosome 1q32-41, and is expressed as a approximately 9 kb transcript in myogenic cells and in adult brain tissue. Forced expression of h-eag in undifferentiated myoblasts generates a current with remarkable similarity to I(K(NI)) indicating that h-eag constitutes the channel responsible for this current in vivo.

Role of an inward rectifier K+ current and of hyperpolarization in human myoblast fusion.

1. The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion-competent myoblasts (FCMBs), and freshly formed myotubes. 2. Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non-inactivating delayed rectifier, and IK(IR), an inward rectifier. 3. IK(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28% of the FCMBs and in all myotubes. 4. IK(IR) is reversibly blocked by Ba2+ or Cs+. 5. Cells expressing IK(IR) have resting membrane potentials of -65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (-32 mV). 6. Cs+ and Ba2+ ions reduce myoblast fusion. 7. It is hypothesized that the IK(IR)-mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T-type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion.

Role of nicotinic acetylcholine receptors at the vertebrate myotendinous junction: a hypothesis.

It has long been known that nicotinic acetycholine receptors (nAChRs) are present in muscle fibres not only at the end plate region but also at the myotendinous junction (MTJ). Their function at the MTJ, however, is yet unknown. Recent experiments in our laboratory lead us to suggest that nAChRs at this site might be involved in muscle repair. MTJ is subject to high mechanical stress and therefore is easily damaged. We found in pure cultures of human myogenic cells that (1) the density of nAChRs in myoblasts increases markedly just before cell fusion, (2) the fusion of human myoblasts is accelerated by the presence of a cholinergic agonist acting on nAChRs and (3) human myoblasts and myotubes spontaneously release an ACh-like compound. Based on these observations we propose that in damaged muscles the nAChRs at the MTJ and those of myogenic cells are activated by the ACh-like compound these cells release. This leads to fusion of myogenic cells with damaged muscle fibres and hence promotes repair.

Contribution of a non-inactivating potassium current to the resting membrane potential of fusion-competent human myoblasts.

1. Using the patch-clamp technique, a new non-inactivating voltage-gated potassium current, IK(ni), was studied in cultured fusion-competent human myoblasts. 2. IK(ni) is activated at voltages above -50 mV and its conductance reaches its maximum around +50 mV. Once activated, the current remains at a steady level for minutes. 3. Reversal potential measurements at various extracellular potassium concentrations indicate that potassium ions are the major charge carriers of IK(ni). 4. IK(ni) is insensitive to potassium channel blockers such as charybdotoxin, dendrotoxins, mast cell degranulating (MCD) peptide, 4-aminopyridine (4-AP), 3,4-diaminopyridine (3,4-DAP) and apamin, but can be blocked by high concentrations of TEA and by Ba2+. 5. A potassium channel of small conductance (8.4 pS at +40 mV) with potential dependence and pharmacological properties corresponding to those of IK(ni) in whole-cell recording is described. 6. IK(ni) participates in the control of the resting potential of fusion-competent myoblasts, suggesting that it may play a key role in the process of myoblast fusion.

Regulation of neuropeptide stoichiometry in neurosecretory cells.

Peptidergic neurons and neurosecretory cells often contain multiple peptides, where they may be present in characteristic ratios. In this article, we describe how a set of five colocalized and coreleased peptides, two adipokinetic hormones (AKH I and AKH II), and three dimeric peptides (APRP 1, 2, and 3) are synthesized by the neurosecretory cells of the corpora cardiaca of the locust Schistocerca gregaria. We show that the five peptides are produced from two prohormones called pro-AKH I, or A-chain, and pro-AKH II, or B-chain. The amino acid sequences as determined by direct protein sequencing are given for both. Prior to processing, the two prohormones form the three possible dimers by the oxidation of the single cysteine residues found in each. The dimers, not the prohormones, are the direct precursors of the peptides. The dimeric precursors are called P1 (A-A), P2 (A-B), and P3 (B-B). Processing results in the generation of the two AKH peptides and the three dimers called adipokinetic hormone precursor-related peptides, or APRPs. Throughout postembryonic development, we show that the ratios of the AKHs and APRPs change dramatically and systematically. We show that these changes can be explained by the differential regulation of the synthesis of the two prohormones and their random association into dimers that are then completely processed. Regulation of peptide stoichiometry may expand the potential information content of the signals generated by multipeptide-producing neurons.

Synthesis of a homodimer neurohormone precursor of locust adipokinetic hormone studied by in vitro translation and cDNA cloning.

The homodimer neurohormone precursor P1, consisting of 41 residue subunits or A-chains, is synthesized by the glandular neurosecretory cells of the corpora cardiaca (CC) of the locust Schistocerca gregaria. Processing of P1 generates two copies of a 10 amino acid peptide neurohormone (AKH I) and one copy of a homodimer peptide (APRP 1). Here we show that the P1 dimer is formed from two independent A-chain translation products. Translation of CC mRNA in vitro produces a prominent 6.4 kd protein, the synthesis of which can be blocked by oligonucleotides hybridizing to mRNA encoding the A-chain. Northern blot experiments suggest that the 6.4 kd protein is produced by an integral of 500 base mRNA. cDNA cloning reveals a pre-A-chain structure in which a single copy of the A-chain is preceded by a 22 amino acid signal peptide. This evidence indicates that the P1 dimer is synthesized by coupling of very small translational products rather than by folding and processing of a larger protein containing more than one copy of the A-chain.