Randomizing the human genome by engineering recombination between repeat elements.

We lack tools to edit DNA sequences at scales necessary to study 99% of the human genome that is noncoding. To address this gap, we applied CRISPR prime editing to insert recombination handles into repetitive sequences, up to 1697 per cell line, which enables generating large-scale deletions, inversions, translocations, and circular DNA. Recombinase induction produced more than 100 stochastic megabase-sized rearrangements in each cell.

The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations.

Mutagenic outcomes of CRISPR/Cas9-generated double-stranded breaks depend on both the sequence flanking the cut and cellular DNA damage repair. The interaction of these features has been largely unexplored, limiting our ability to understand and manipulate the outcomes. Here, we measured how the absence of 18 repair genes changed frequencies of 83,680 unique mutational outcomes generated by Cas9 double-stranded breaks at 2,838 synthetic target sequences in mouse embryonic stem cells.

Prediction of prime editing insertion efficiencies using sequence features and DNA repair determinants.

Most short sequences can be precisely written into a selected genomic target using prime editing; however, it remains unclear what factors govern insertion. We design a library of 3,604 sequences of various lengths and measure the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems in varying DNA repair contexts. We find that length, nucleotide composition and secondary structure of the insertion sequence all affect insertion rates.

Predicting base editing outcomes using position-specific sequence determinants.

CRISPR/Cas base editors promise nucleotide-level control over DNA sequences, but the determinants of their activity remain incompletely understood. We measured base editing frequencies in two human cell lines for two cytosine and two adenine base editors at ∼14 000 target sequences and find that base editing activity is sequence-biased, with largest effects from nucleotides flanking the target base. Whether a base is edited depends strongly on the combination of its position in the target and the preceding base, acting to widen or narrow the effective editing window.

Effective lung-targeted RNAi in mice with peptide-based delivery of nucleic acid.

We have previously developed efficient peptide-based nucleic acid delivery vectors PF14 and NF55, where we have shown that these vectors preferentially transfect lung tissue upon systemic administration with the nucleic acid. In the current work, we have explored the utilization and potential of these vectors for the lung-targeted gene therapy. Accordingly, we assessed the efficacy of these peptides in (i) two different lung disease models - acute lung inflammation and asthma in mice and (ii) using two different nucleic acid cargos - siRNA and pDNA encoding shRNA.

Effective in vivo gene delivery with reduced toxicity, achieved by charge and fatty acid -modified cell penetrating peptide.

Non-viral gene delivery systems have gained considerable attention as a promising alternative to viral delivery to treat diseases associated with aberrant gene expression. However, regardless of extensive research, only a little is known about the parameters that underline in vivo use of the nanoparticle-based delivery vectors. The modest efficacy and low safety of non-viral delivery are the two central issues that need to be addressed.

Treatment of cutaneous squamous cell carcinomas by intralesional interferon alfa-2b therapy.

Background And Design: Intralesional recombinant interferon alfa-2b has been shown to be effective in the treatment of actinic keratoses and basal cell carcinomas. This open-label study was designed to evaluate the effectiveness and cosmetic result of this therapy on actinically induced, primary cutaneous squamous cell carcinomas. Thirty-six squamous cell carcinomas (28 invasive lesions and 8 in situ lesions) ranging in size from 0.

Once-daily 0.1% mometasone furoate cream versus twice-daily 0.1% betamethasone valerate cream in the treatment of a variety of dermatoses.

A randomized, investigator-blind, parallel-group trial was conducted to compare the safety and efficacy of 0.1% mometasone furoate cream applied once daily with that of 0.1% betamethasone valerate cream applied twice daily in patients (n = 69) with allergic contact dermatitis, atopic dermatitis and other steroid-responsive dermatoses.

Intralesional interferon therapy for basal cell carcinoma.

In a clinical trial of 172 patients at four medical centers, interferon alfa-2b (1.5 x 10(6) IU) or a placebo was injected directly into biopsy-proved noduloulcerative or superficial basal cell carcinomas three times weekly for 3 weeks, for a cumulative dose of 13.5 million IU.

The effect of an intralesional sustained-release formulation of interferon alfa-2b on basal cell carcinomas.

Intralesional interferon alfa-2b has been proven effective in the treatment of basal cell carcinomas. Because nine injections over a 3-week period have been necessary to produce clinically significant cure rates, a sustained-release protamine zinc chelate interferon formulation has been developed. In this study, 65 basal cell carcinomas were treated in one of two dosing schedules with intralesional sustained release interferon alfa-2b (10 million IU per injection).

A randomized trial of combination therapy with intralesional interferon alpha 2b and podophyllin versus podophyllin alone for the therapy of anogenital warts.

To determine the value of combining interferon with standard local therapy in the treatment of human papillomavirus infection, 97 patients with anogenital warts were randomized to a short course of either interferon plus podophyllin or podophyllin alone. Interferon alpha 2b (1.5 x 10(6) IU) was injected intralesionally and podophyllin resin applied topically to each of three warts once weekly for 3 weeks.

Intralesional interferon alfa-2b for the treatment of genital warts.

A randomized, double-blind, placebo-controlled study has been conducted, and intralesional interferon alfa-2b was tested in the treatment of genital warts. This study design was to give 1 million units interferon alfa-2b intralesionally into the base of each of five external genital warts per patient, on a Monday-Wednesday-Friday treatment schedule for 3 weeks (total of nine injections). Forty-two patients were entered (20 randomized to receive interferon and 22 placebo injections).

Treatment of basal cell carcinoma with intralesional interferon.

Eight patients with basal cell carcinomas were treated with recombinant alpha-2 interferon. Each patient had a biopsy-proved basal cell carcinoma of the nodular or superficial type that was injected intralesionally three times a week for 3 weeks (9 total injections) with 1.5 X 10(6) IU (0.

Sulfhydryl reactivity: mechanism of action of several antiviral compounds--selenocystine, 4-(2-propinyloxy)-beta-nitrostyrene, and acetylaranotin.

The addition of 5 mM dithiothreitol to a cell-free assay system for influenza ribonucleic acid (RNA) polymerase activity reverses the inhibitory activity otherwise possessed by three established antiviral compounds: selenocystine, 4-(2-propinyloxy)-beta-nitrostyrene, and acetylaranotin. Although 50% or greater enzyme inhibitory activity is repeatedly achieved for these compounds at a concentration of approximately 50 mug/ml (0.1 to 0.