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Researchers Submit Patent Application, "Modulation of Bio-Electrical Rhythms via a Novel Engineering Approach", for Approval

August 7, 2014

By a News Reporter-Staff News Editor at Gene Therapy Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors Marban, Eduardo (Lutherville, MD); Li, Ronald A. (Baltimore, MD); Tsang, Suk-Ying (Shatin, HK); Cho, Heecheol (Columbia, MD); Xue, Tian (Baltimore, MD), filed on January 21, 2014, was made available online on July 24, 2014 (see also The Johns Hopkins University).

The patent's assignee is The Johns Hopkins University.

News editors obtained the following quote from the background information supplied by the inventors: "Spontaneous cellular electrical rhythms (or pacing) govern numerous biological processes from the autonomous beating of the heart, pain transmission, to respiratory rhythms and insulin secretion. For instance, spontaneous neuronal electrical discharges in damaged dorsal root ganglions underlie neuropathic pain but limited useful therapy is available. Recently, we have also discovered that key ionic components which underlie the electrical rhythms in pancreatic .beta. cells also modulate the secretion of insulin. In the heart, abnormal pacing leads to various forms of arrhythmias and electrical disorders that necessitate traditional pharmacologic interventions and implantation of costly electronic devices that are associated with various side effects, inherent risks, and expenses.

"Autonomous rhythmic heart beats are modulated by sympathetic and parasympathetic means according to everyday needs; such normal rhythms originate in the sino-atrial (SA) node of the heart, a specialized cardiac tissue consisting of a few thousands pacemaker cells that spontaneously generate rhythmic action potentials (AP) (i.e. pacing). One of the key players known to prominently modulate the pacing activity of SA nodal cells is the cardiac membrane current I.sub.f ('f' for funny), a depolarizing, mixed Na.sup.+/K.sup.+ inward current.sup.1. Despite the fact that I.sub.f has been recognized for over 20 years, the encoding genes, collectively known as the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channel gene family, have been cloned relatively recently..sup.2-4 To date, four isoforms, namely HCN1-4, each with a distinct pattern of tissue distribution and biophysical profiles, have been identified.sup.3-8. Of the two predominant isoforms in the SA node, time-dependent HCN1 currents open .about.40 times faster than those of HCN4 channels.sup.9-13; a single base-pair deletion mutation detected in the human HCN4 gene of a patient has been linked to idiopathic sinus node dysfunction.sup.14.

"Since HCN1-4 readily co-assemble to form heterotetrameric complexes with context-dependent properties that can not be predicted from the individual isoforms.sup.15-18, native I.sub.f can have complex molecular identity depending upon the particular isoforms expressed. Furthermore, HCN channels activate more positively in native cardiomyocytes than in mammalian expression systems, suggesting that the gating properties of I.sub.f are highly context-dependent (Qu et al 2002 Pflugers) (e.g. the presence of endogenous subunits). Thus, native I.sub.f is difficult to reproduce by simple expression of a single HCN isoform. Indeed, previous attempts to overexpress wild-type HCN2 in adult ventricular cardiomyocytes failed to induce automaticity.sup.19, presumably due to its negative activation relative to the voltage range of cardiac pacing. It would be highly desirable to develop a flexible and effective approach that enables us to delicately customize the activity of HCN channels so as to achieve a range of therapeutic outcomes. For instance, to engineer a HCN channel construct, which opens more readily than wild-type HCN channels (and thereby compensates the context-dependent negative activation shift in heart cells) to better mimic native nodal I.sub.f so as to effectively induce or modulate cardiac automaticity. The same principle can be extrapolated for application in other cell types whose functions depend on electrical rhythms.

"As previously mentioned, HCN-encoded I.sub.f (or I.sub.h) plays an important role in the spontaneous rhythmic activity in cardiac, neuronal as well as insulin-secreting cells (32-38). Although classical depolarization-activated voltage-gated K.sup.+ (K.sub.v) and HCN channels are structurally homologous to each other, the latter are uniquely distinctive from the K.sub.v counterparts by their signature 'backward' gating (i.e. activation upon hyperpolarization rather than depolarization). The basis of HCN gating is largely unknown.

"Recent evidence suggests that the voltage-sensing mechanisms of HCN and K.sub.v channels are conserved despite their opposite gating behaviors (i.e. the HCN S4 also moves outward and inward during depolarization and hyperpolarization, respectively) (39,40). This finding raises the possibility that the S3-S4 linker (defined as residues 229EKGMDSEVY237 of HCN1; FIG. 1), which is directly tethered to the S4 voltage-sensor, also influences the activation phenotypes of HCN channels as does that of K.sub.v channels (41-43). Indeed, we have recently reported that the S3-S4 linker contains several functionally-important residues (44, 45). For instance, single alanine substitutions of G213, M232 and E235 produced depolarizing activation shifts. The pattern of site-dependent perturbations of HCN activation, along with computational modeling, further suggests that part of the linker conforms a helical secondary structure with the determinants G231, M232 and E235 clustered on one side. It would be desirable to understand the structural and functional roles of the HCN S3-S4 linker. Such understanding would be helpful in developing engineered HCN channels which would open more (or less) readily for increasing (or decreasing) automaticity in cardiac, neuronal, pancreatic cells, etc.

"Throughout this application, various publications are referenced to by numbers. Full citations for these publications may be found at the end of the specification immediately following the Abstract. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to those skilled therein as of the date of the invention described and claimed herein."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "Here we have conceived of a novel method to modulate or 'custom-tailor' bio-electrical rhythms in such specialized cells as cardiac, neuronal and pancreatic cells, by targeting the activity of particular ion channels using a novel protein- and genetic-engineering combined approach. In combination with protein engineering, this can be accomplished by in vivo or ex vivo gene transfer of specific normal and/or engineered ion channel proteins into native tissues or stem cell-derived derivatives (followed by cell transplantation), respectively, to produce the desired physiological consequences in vivo. For instance, one can first engineer HCN channels such that their activation thresholds are shifted either above or below that of normal channels by a given level. Such genetically engineered recombinant constructs, when introduced into cells, can alter the associated bio-electrical rhythmic activity and subsequently augment or attenuate their physiological responses (e.g. heart beat, the feeling of pain, appetite, insulin secretion, etc). Collectively, our approach may lead to new effective therapies for such clinical problems as arrhythmias (sick sinus syndrome); epilepsy, neuropathic pain, obesity, diabetes, etc. The efficacy of this approach has been verified by our in vivo gene transfer experiments, in the context of the heart, to correct an arrhythmias (sick sinus syndrome) described in the paragraphs that follow.

"We now provide gene transfer and cell administration methods that can create a 'bio-battery' to exert pacemaking function, and/or to modulate the activity of an endogenous or induced cardiac pacemaker function using genetically modified HCN constructs.

"Methods of the invention using genetically modified HCN constructs may be employed to create and/or modulate the rhythmic activity of an endogenous pacemaker (such as the sinotrial node of a mammalian heart, neuronal cells of the central nervous system, .beta.-cells of the pancreas, etc) and/or an induced pacemaker (e.g. biological pacemaker generated from stem cells or converted electrically-quiescent cells).

"More particularly, in a preferred aspect of the invention, quiescent heart muscle cells are converted into pacemaker cells by in vivo viral gene transfer or modified cell transfer (e.g., differentiated stem cells).

"In a further aspect, a composition is administered to a subject to alter the frequency of (i.e. to tune) an existing endogenous or induced cardiac pacemaker function. Polynucleotides of or modified cells containing a genetically modified HCN construct that displays a particular phenotype are preferred agents for administration. The method can be applied to virtually any cell types that exhibit bioelectrical rhythms (e.g. certain neuronal and pancreatic cells) other than cardiac cells so as to subsequently modify/improve their physiological functions. For instance, the activity of electrically active neuronal cells in the case of neuropathic pain can be decreased using a different HCN construct (e.g. one that is less ready for opening or even a dominant-negative construct) to reduce or eliminate the sensation of pain. (i.e. to correct a particular defect due to diseases or traumas).

"Specifically, the present application discloses that the length of the HCN1 S3-S4 linker is a determinant of gating. Systematic alteration of the linker length by deletion and insertion, followed by detailed characterization of the associated functional consequences, revealed that the linker length and its amino acid constituents prominently modulate gating properties in a systematic pattern. However, the major principle is that other functional HCN domains (e.g. S4 and the pore region that affect gating properties, the cAMP binding domain, etc) can be similarly modified to achieve a particular outcome.

"Other aspects of the invention are discussed infra.


"FIG. 1 represents illustrations of the putative transmembrane topology of HCN1 wherein A) The six putative transmembrane segments (S1-S6) of a monomeric HCN1 subunit. The S3-S4 linker is thickened; B) S3-S4 linker sequences of WT HCN1 and other channel constructs investigated in the present study. Functional and non-functional constructs are labeled green and purple, respectively. M232 is highlighted in red.

"FIG. 2 represents illustrations of the effects of shortening the S3-S4 linker on HCN1 currents; Representative traces of whole-cell currents recorded from cells expressing WT and different deletion HCN1 constructs. .DELTA.229-231, .DELTA.233-237, .DELTA.234-237, .DELTA.235-237, .DELTA.229-231/.DELTA.233-237, .DELTA.229-231/.DELTA.234-237 and .DELTA.229-231/.DELTA.235-237 expressed robust hyperpolarization-activated time-dependent currents. In contrast, oocytes injected with .DELTA.229-234, .DELTA.229-237, .DELTA.232-234, and .DELTA.232-237 produced no measurable currents.

"FIG. 3 represents illustrations of the steady-state I-V relationships of S3-S4 deletion constructs. The steady-state current-voltage (I-V) relationship was determined by plotting the HCN1 currents measured at the end of the 3-second pulse. Inset, Electrophysiological protocol used to elicit currents. Data shown are mean.+-.SEM.

"FIG. 4 represents illustrations of the effects of S3-S4 linker deletions on HCN1 steady-state activation. A) Representative tail currents through WT, .DELTA.229-231/.DELTA.233-237, .DELTA.229-231/.DELTA.234-237, .DELTA.229-231/.DELTA.235-237 and .DELTA.229-231 (a, -120 mV; b, -100 mV; c, -80mV) normalized to the maximum current recorded. B) Steady-state activation curves for WT, .DELTA.229-231/233-237, .DELTA.229-231/234-237, .DELTA.229-231/235-237 and .DELTA.229-231. Increasing the S3-S4 linker length as observed from these constructs, caused sequential depolarizing activation shifts (also see FIG. 8).

"FIG. 5. represents illustrations of the effects of prolonging the S3-S4 linker with glutamines on HCN1 steady-state activation. A) Representative traces of whole-cell currents and their corresponding tail currents recorded from WT, InsQ233Q, InsQQ233QQ, InsQQQ233QQQ and 237InsQQQ channels. a, -120 mV; b, -100 mV; c, -80 mV. B) Steady-state activation curves of the same channels shown in A). Extensive prolongation of the S3-S4 linker shifted activation negatively (see text and FIG. 8).

"FIG. 6 represents illustrations of the effects of prolonging the S3-S4 linker (i.e. Dup229-232 & Dup229-237) on HCN1 activation. A) Representative whole-cell currents and their corresponding tail currents through WT, Dup229-232 and Dup229-237 channels (a, -120 mV; b, -100 mV; c, -80 mV) normalized to the maximum current recorded.

"B) Steady-state activation curves. Consistent with the glutamine insertion constructs, the steady-state activation curves of the duplication constructs were also negative shifted. Activation curve of Dup229-237 was more hyperpolarized than that of Dup229-232.

"FIG. 7 represents illustrations of the effects of prolonging (top) and shortening (bottom) the S3-S4 linker on activation (.tau..sub.act) (A) and deactivation (.tau..sub.deact) (B) kinetics. The electrophysiological protocols used for inducing activation and deactivation are displayed.

"FIG. 8 is a graphic representation summarizing the effects of the S3-S4 linker length on HCN1 steady-state activation. A strong correlation was observed between the V.sub.1/2 of the channels and their linker length.

"FIG. 9, A thru H are representative illustrations of the effects of HCN2 overexpression in cardiomyoctyes.

"FIG. 10, A and B are representative illustrations demonstrating the effects of I.sub.K1 suppression.

"FIG. 11, A thru D are representative illustrations demonstrating, in part, that modified HCN1 can functionally substitute as an electronic device in vivo.

"FIG. 12, A thru D are representative illustrations demonstrating, in part, that modified HCN1 can functionally substitute as an electronic device in vivo."

For additional information on this patent application, see: Marban, Eduardo; Li, Ronald A.; Tsang, Suk-Ying; Cho, Heecheol; Xue, Tian. Modulation of Bio-Electrical Rhythms via a Novel Engineering Approach. Filed January 21, 2014 and posted July 24, 2014. Patent URL:

Keywords for this news article include: Pain, Genetics, Pancreas, Arrhythmia, Cardiology, Neuropathy, Pacemakers, Proinsulin, Engineering, Cardio Device, Cardiomyocyte, Medical Devices, Gastroenterology, Peptide Hormones, Nervous System Diseases, Neurologic Manifestations, The Johns Hopkins University.

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Source: Gene Therapy Weekly

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