Severe peripheral nerve injuries have devastating consequences on the quality of life in affected patients, and they represent a significant unmet medical need. Destruction of nerve fibers results in denervation of targeted muscles, which, subsequently, undergo progressive atrophy and loss of function. Timely restoration of neural innervation to muscle fibers is crucial to the preservation of muscle homeostasis and function. The goal of this study was to evaluate the impact of addition of adipose stem cells (ASCs) to polycaprolactone (PCL) nerve conduit guides on peripheral nerve repair and functional muscle recovery in the setting of a critical size nerve defect. To this end, peripheral nerve injury was created by surgically ablating 6 mm of the common peroneal nerve in a rat model. A PCL nerve guide, filled with ASCs and/or poloxamer hydrogel, was sutured to the nerve ends. Negative and positive controls included nerve ablation only (no repair), and reversed polarity autograft nerve implant, respectively. Tibialis anterior (TA) muscle function was assessed at 4, 8, and 12 weeks postinjury, and nerve and muscle tissue was retrieved at the 12-week terminal time point. Inclusion of ASCs in the PCL nerve guide elicited statistically significant time-dependent increases in functional recovery (contraction) after denervation; *25% higher than observed in acellular (poloxamer-filled) implants and indistinguishable from autograft implants, respectively, at 12 weeks postinjury (p < 0.05, n = 7-8 in each group). Analysis of single muscle fiber cross-sectional area (CSA) revealed that ASC-based treatment of nerve injury provided a better recapitulation of the overall distribution of muscle fiber CSAs observed in the contralateral TA muscle of uninjured limbs. In addition, the presence of ASCs was associated with improved features of re-innervation distal to the defect, with respect to neurofilament and S100 (Schwann cell marker) expression. In conclusion, these initial studies indicate significant benefits of inclusion of ASCs to the rate and magnitude of both peripheral nerve regeneration and functional recovery of muscle contraction, to levels equivalent to autograft implantation. These findings have important implications to improved nerve repair, and they provide input for future work directed to restoration of nerve and muscle function after polytraumatic injury.

Population-level control of HIV-1 faces recognized challenges, including the evolution of viral resistance and adherence issues in resource-limited settings. It has long beenproposed that viral deletion mutants that conditionally self-renew at the expense of the wild-type virus (i.e., Defective Interfering Particles, DIPs 1 ) could constitute a longterm intervention that circumvents adherence challenges and has a high genetic barrier to resistance, echoing recent approaches 2 . Theories predict 3,4 that DIPs could be engineered into a therapy for HIV-1 (i.e., Therapeutic interfering particles or 'TIPs') provided they stably persist in patients (R0>1) by spreading to new cells during active infection (hence, a self-replenishing antiviral). To date, DIPs amenable to such engineering have remained elusive for HIV-1. Here we report the discovery of an HIV-1 DIP and its subsequent engineering into a TIP. The TIP interferes with HIV-1 replication at multiple stages of the viral lifecycle, including genome packaging, virion maturation, and reverse transcription, essentially acting as a combination antiviral.In humanized mice, the TIP suppressed HIV-1 replication by ten-fold and significantly protected CD4+ T cells from HIV-1 mediated depletion. These data provide proof-ofconcept for a class of biologic with the potential to circumvent significant barriers to HIV-1 control.Despite the availability of effective antiretroviral therapy regimens, 37 million people are currently living with HIV-1/AIDS (hereafter HIV) and ~2 million new HIV infections occur each year, largely in the developing world 5 . Epidemiological models 6 project that epidemic control in sub-Saharan Africa using conventional approaches may be exceptionally

AbstractIt has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare, spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback, and then barcoded for tracking via sequencing (i.e., Random-Deletion Library sequencing, RanDeL-seq). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1’s cis and trans elements. The resulting landscape recapitulated HIV-1’s known cis-acting elements (i.e., LTR, Ψ, and RRE) and surprisingly indicated that HIV-1’s central DNA flap (i.e., central polypurine tract, cPPT to central termination sequence, CTS) is as critical as the LTR, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼300bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV’s cis-acting regions are larger than the UTR termini, encompassing a large fraction of the non-structural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections.ImportanceRecent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)—a subset of viral deletion mutant that conditionally replicate. Identifying and engineering DIPs requires that viral cis- and trans-acting elements be accurately mapped. Here we introduce a high-throughput method (Random Deletion Library sequencing, RanDeL-seq) to comprehensively map cis- and trans-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central poly-purine tract (cPPT) and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.

It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes, but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback and then barcoded for tracking via sequencing (i.e., random deletion library sequencing [RanDeL-seq]). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1’s cis and trans elements. The resulting landscape recapitulated HIV-1’s known cis-acting elements (i.e., long terminal repeat [LTR], Ψ, and Rev response element [RRE]) and, surprisingly, indicated that HIV-1’s central DNA flap (i.e., central polypurine tract [cPPT] to central termination sequence [CTS]) is as critical as the LTR, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼300-bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV’s cis-acting regions are larger than the untranscribed (UTR) termini, encompassing a large fraction of the nonstructural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants, enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections. IMPORTANCE Recent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)—a subset of viral deletion mutants that conditionally replicate. Identifying and engineering DIPs require that viral cis- and trans-acting elements be accurately mapped. Here, we introduce a high-throughput method (random deletion library sequencing [RanDeL-seq]) to comprehensively map cis- and trans-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central polypurine tract (cPPT), and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.

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Abbreviations: circuit disrupting oligonucleotide therapy (C-DOT); human cytomegalovirus (CMV); herpes simplex virus type 1 (HSV-1) Abstract: 222 words Main Text: 1978 words Figures: 4 (color) Abstract:From bacteria to cancers, it has long been recognized that drug-resistant mutants emerge quickly, causing significant morbidity and mortality 1-3 . Antiviral resistance in herpesviruses is of particular concern 4,5 with herpes simplex virus type 1 (HSV-1)-a leading cause of blindness-and human herpesvirus 5, cytomegalovirus (CMV)-a leading cause of birth defects and transplant failureexhibiting substantial resistance to standard-of-care antivirals in the clinic 6,7 . Combination therapies, which limit resistance by necessitating multiple viral mutations, can be effective but increase the risk of off-target effects and associated toxicity and are absent for most viral diseases.Here, we present proof-of-concept for a novel approach that disrupts viral auto-regulatory circuits with a single molecule and limits resistance by requiring multiple viral mutations. We develop DNA-based circuit-disruptor oligonucleotide therapies (C-DOTs) that exploit this mechanism by interfering with transcriptional negative feedback in human herpesviruses (CMV and HSV-1) thereby increasing viral transcription factors to cytotoxic levels. C-DOTs reduce viral replication >100-fold, prevent emergence of resistant mutants in continuous culture, are effective in highviremic conditions where existing antivirals are ineffective, and show efficacy in mice. Strikingly, no C-DOT-resistant mutants evolved in >60 days of culture, in contrast to approved herpesvirus antivirals where resistance rapidly evolved. Overall, the results demonstrate that oligonucleotide therapies targeting feedback circuits are escape resistant and could have broad therapeutic applicability to viruses, microbes, and neoplastic cells. Main text:The time to emergence of drug-resistant 'escape' mutants is typically estimated from the mutation rate, µ, and the effective population size, N 8,9 . Many viruses exhibit large µ such that frequency of mutants (µ´N) is >1, even for moderate virus population sizes (e.g., if µ~10 -5 then for µ´N > 1 requires only that N > 10 5 ). Herpesviruses, for example, exhibit high mutation rates 10,11 , which may explain the substantial antiviral resistance observed in clinical settings 4,5 . In particular, herpes simplex virus type 1 (HSV-1)-a leading cause of blindness-exhibits resistance to acyclovir (ACV) in ~40% of transplant patients 6 while human herpesvirus 5, cytomegalovirus (CMV)-a leading cause of birth defects and transplant failure-exhibits resistance to ganciclovir (GCV) in 30-75% of patients 7 . ACV and GCV resistance arises because their antiviral activity requires herpesvirus thymidine kinase (TK), and single-base mutations destroy TK activity (with a µ ~10 -3 ) 10 driving TK escape mutants within a single generation 12 , which ultimately led to the development of non-TK drug targets 13,14 . Resistance to these new therapies is sti...

Techniques by which to genetically manipulate members of the microbiota enable both the evaluation of host-microbe interactions and an avenue by which to monitor and modulate human physiology. Genetic engineering applications have traditionally focused on model gut residents, such as Escherichia coli and lactic acid bacteria.

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