Phenotypes Associated With the Val122Ile, Leu58His, and Late-Onset Val30Met Variants in Patients With Hereditary Transthyretin Amyloidosis.

BACKGROUND AND OBJECTIVES: Hereditary transthyretin amyloidosis (hATTR) is a rare autosomal dominant systemic disease with variable penetrance and heterogeneous clinical presentation. Several effective treatments can reduce mortality and disability, though diagnosis remains challenging, especially in the United States where disease is nonendemic. Our aim is to describe the neurologic and cardiac characteristics of common US ATTR variants V122I, L58H, and late-onset V30M at presentation. METHODS: We conducted a retrospective case series of patients with a new diagnosis of ATTRv between January 2008 and January 2020 to characterize features of prominent US variants. The neurologic (examination, EMG, and skin biopsy), cardiac (echo), and laboratory assessments (pro b-type natriuretic peptide [proBNP] and reversible neuropathy screens) are described. RESULTS: A total of 56 patients with treatment-naïve ATTRv with symptoms/signs of peripheral neuropathy (PN) or cardiomyopathy and confirmatory genetic testing presenting with Val122Ile (N = 31), late-onset Val30Met (N = 12), and Leu58His ATTRv (N = 13) were included. The age at onset and sex distributions were similar (V122I: 71.5 ± 8.0, V30M: 64.8 ± 2.6, and L58H: 62.4 ± 9.8 years; 26, 25, 31% female). Only 10% of patients with V122I and 17% of patients with V30M were aware of an ATTRv family history, while 69% of patients with L58H were aware. PN was present in all 3 variants at diagnosis (90%, 100%, and 100%), though neurologic impairment scores differed: V122I: 22 ± 16, V30M: 61 ± 31, and L58H: 57 ± 25. Most points (deficits) were attributed to loss of strength. Carpal tunnel syndrome (CTS) and a positive Romberg sign were common across all groups (V122I: 97%, 39%; V30M: 58%, 58%; and L58H: 77%, 77%). ProBNP levels and interventricular septum thickness were highest among patients with V122I (5,939 ± 962 pg/mL, 1.70 ± 0.29 cm), followed by V30M (796 ± 970 pg/mL, 1.42 ± 0.38 cm) and L58H (404 ± 677 pg/mL, 1.23 ± 0.36 cm). Atrial fibrillation was present among 39% of cases with V122I and only 8% of cases with V30M and L58H. Gastrointestinal symptoms were rare (6%) among patients with V122I and common in patients with V30M (42%) and L58H (54%). DISCUSSION: Important clinical differences exist between ATTRv genotypes. While V122I is perceived to be a cardiac disease, PN is common and clinically relevant. Most patients with V30M and V122I were diagnosed de novo and therefore require clinical suspicion for diagnosis. A history of CTS and a positive Romberg sign are helpful diagnostic clues.

Mycobacterium leprae induces Schwann cell proliferation and migration in a denervated milieu following intracutaneous excision axotomy in nine-banded armadillos.

Nine-banded armadillos develop peripheral neuropathy after experimental Mycobacterium leprae infection that recapitulates human disease. We used an intracutaneous excision axotomy model to assess the effect of infection duration by M. leprae on axonal sprouting and Schwan cell density. 34 armadillos (17 naïve and 17 M. leprae-infected) underwent 3 mm skin biopsies to create an intracutaneous excision axotomy followed by a concentric 4-mm overlapping biopsy 3 and 12-months post M. leprae inoculation. A traditional distal leg biopsy was obtained at 15mo for intraepidermal nerve fiber (IENF) density. Serial skin sections were immunostained against a axons (PGP9.5, GAP43), and Schwann cells (p75, s100) to visualize regenerating nerves. Regenerative axons and proliferation of Schwann cells was measured and the rate of growth at each time point was assessed. Increasing anti-PGL antibody titers and intraneural M. leprae confirmed infection. 15mo following infection, there was evidence of axon loss with reduced distal leg IENF versus naïve armadillos, p < 0.05. This was associated with an increase in Schwann cell density (11,062 ± 2905 vs. 7561 ± 2715 cells/mm3, p < 0.01). Following excisional biopsy epidermal reinnervation increased monotonically at 30, 60 and 90 days; the regeneration rate was highest at 30 days, and decreased at 60 and 90 days. The reinnervation rate was highest among animals infected for 3mo vs those infected for 12mo or naïve animals (mean ± SD, 27.8 ± 7.2 vs.16.2 ± 5.8vs. 15.3 ± 6.5 mm/mm3, p < 0.05). The infected armadillos displayed a sustained Schwann cell proliferation across axotomy time points and duration of infection (3mo:182 ± 26, 12mo: 256 ± 126, naive: 139 ± 49 cells/day, p < 0.05). M. leprae infection is associated with sustained Schwann cell proliferation and distal limb nerve fiber loss. Rates of epidermal reinnervation were highest 3mo after infection and normalized by 12 mo of infection. We postulate that excess Schwann cell proliferation is the main pathogenic process and is deleterious to sensory axons. There is a compensatory initial increase in regeneration rates that may be an attempt to compensate for the injury, but it is not sustained and eventually followed by axon loss. Aberrant Schwann cell proliferation may be a novel therapeutic target to interrupt the pathogenic cascade of M. leprae.

Photoactivation of sulfonated polyplexes enables localized gene silencing by DsiRNA in breast cancer cells.

Translation potential of RNA interference nanotherapeutics remains challenging due to in vivo off-target effects and poor endosomal escape. Here, we developed novel polyplexes for controlled intracellular delivery of dicer substrate siRNA, using a light activation approach. Sulfonated polyethylenimines covalently linked to pyropheophorbide-α for photoactivation and bearing modified amines (sulfo-pyro-PEI) for regulated endosomal escape were investigated. Gene knock-down by the polymer-complexed DsiRNA duplexes (siRNA-NPs) was monitored in breast cancer cells. Surprisingly, sulfo-pyro-PEI/siRNA-NPs failed to downregulate the PLK1 or eGFP proteins. However, photoactivation of these cell associated-polyplexes with a 661-nm laser clearly restored knock-down of both proteins. In contrast, protein down-regulation by non-sulfonated pyro-PEI/siRNA-NPs occurred without any laser treatments, indicating cytoplasmic disposition of DsiRNA followed a common intracellular release mechanism. Therefore, sulfonated pyro-PEI holds potential as a unique trap and release light-controlled delivery platform for on-demand gene silencing bearing minimal off target effects.

Oxime Ether Lipids as Transfection Agents: Assembly and Complexation with siRNA.

RNAi-based therapeutic approaches to combat cancer and other diseases are currently an area of great interest. However, practical applications of this approach rely on optimal tools to carry and deliver siRNA to the desired site. Oxime ether lipids (OELs) are a class of molecules among other various carriers being examined for siRNA delivery. OELs, relatively new candidates, belong to a class of non-glycerol based lipids and have begun to claim their place as an siRNA delivery carrier in the field of RNAi therapy. Chemical synthesis steps of OELs are considered relatively simple with the ability to modify the functionalities as desired. OEL-siRNA complexes can be assembled in the presence of serum-containing buffers (or cell culture media) and recent data from our and other groups have demonstrated that OELs are viable carriers for siRNA delivery in the cell culture systems. In this chapter, we provide the details of experimental protocols routinely used in our laboratory to examine OEL-siRNA complexes including their assembly, stability, and transfection efficiencies.