Integration-deficient Lentiviral Vector Service

Integration-deficient lentiviral vectors wielding a catalytically dead IN(D64V) mutation behave like molecular shooting stars—brilliantly illuminating transcriptomes yet refusing to leave permanent craters in the host genome. The platform marries a high-capacity HIV-1 backbone with an integrase active-site blockade and a cPPT/CTS hyper-loop, forging a non-insertional shuttle that delivers > 9 kb transcription units as stable episomes without ever committing a base pair to chromatin. Recent protein origami on the integrase surface—through loop shortening, glycan fog deployment, and electrostatic stealth—broadens the nuclear import radius to quiescent stem cells and terminally differentiated neurons, while erasing any residual strand-transfer whispers. When these genomically shy particles are packaged with high-level, detargeted components (bearing codon-optimized gag-pol, SIN LTRs, and lineage-specific miR masking cassettes), the progeny achieves supranuclear titers, negligible RCL background noise, and episomal landscapes that blaze within target nuclei yet vanish like vapor upon cell division.

Fig. 1 Targeting approaches for lentiviral vector pseudotyping^1,2

The governing axiom we uphold is that durable gene expression need not equate to permanent genomic scar tissue. Creative Biolabs's integration-deficient lentiviral service fuses deep knowledge of episome biology, cryo-EM-guided active-site mutagenesis, and single-cell nuclear import bar-coding into a seamless pipeline that dissolves every barrier between your transcriptomic question and a sequence-verified, IN(D64V)-pseudotyped episomal vector. Using serum-free, suspension packaging lines optimized for D64V integrase expression, we deploy combinatorial cPPT/CTS libraries, glycan-shield matrices, and charge-inversion nanosurgery to craft particles with supranuclear titers, negligible RCL background, and pH-responsive fusion kinetics that trigger episome formation only upon target engagement. These integration-deficient vectors, delivered endotoxin- and aggregate-free, illuminate target-positive cells in patient-derived organoids and orthotopic xenografts with cinematic brightness, enabling mechanistic studies to sprint from bench to publication-ready datasets without genomic footprint or procedural delay.

Service Workflow – Phase-gated

The integration-deficient lentiviral platform unites high-level, serum-free suspension producer clones with catalytically dead IN(D64V) codon-optimized gag-pol, cPPT/CTS hyper-loop cassettes, and tandem anion-exchange plus size-exclusion chromatography to deliver endotoxin-undetectable, integration-incompetent lentiviral lots carrying SIN-LTR, miR-masked, or destabilization-domain payloads—ready for immediate transduction of quiescent neural stem cells, hypoxic tumoroids, or BSL-2-permissible primary organoids without genomic insertion, silencing, or cell-cycle bias.

End-to-End Service Modules

Vector Design & Strategy

By treating every kilobase as a design opportunity rather than a constraint, we deliver vectors that align expression strength, cell-type specificity, and safety profile with the unique pharmacology of your therapeutic program.

• Promoter Architecture

We shortlist promoters not from a catalogue but from your biological question. Require ubiquitous expression in hematopoietic lineages? The elongation-factor-1α core plus proximal intron is cloned in its shortest active form to preserve packaging capacity. Prefer a single copy of the chimeric CMV–chicken β-actin enhancer for rapid, high-level onset in dividing T cells? We embed a 5′-UTR Kozak variant that boosts translation without extra bases. For liver-restricted expression we replace the distal enhancer of the human albumin promoter with a 120-bp synthetic module that retains hepatocyte specificity yet withstands methylation silencing. Effective neuronal targeting with a small 0.7-kb Syn1 promoter region with a chromatin-opening element results in strong, cell-type specific expression in mature neurons and minimal leakiness in off-target glia. All promoter fragments were de-novo synthesized, selected to avoid cryptic splice sites, and checked for overlap of transcription-factor footprints to reduce baseline noise.

• Payload Engineering up to 9 kb

Large or complex cassettes are recoded through in-silico algorithms that balance codon usage, GC content, and cryptic splice suppression while preserving protein function. Polycistronic arrangements are assembled with strain-reducing linkers: P2A for equimolar stoichiometry, T2A when rapid cleavage is desired, or EMCV-IRES when differential expression levels are beneficial. Intragenic microRNA sponges are inserted into artificial introns flanked by splice donors/acceptors optimized for the human context. This places them under the same promoter without additional length. Conditional degron tags (auxin- or Shield-1-responsive) are appended at the 3′ end with flexible glycine-serine spacers to avoid steric hindrance, enabling post-translational control of your transgene.

Molecular Construction – From Concept to Sequence

We accept any starting point you choose to provide: a simple text file containing the digital sequence of your novel cassette, an existing plasmid harvested from your freezer stock, or even microgram quantities of in-vitro-transcribed mRNA that encodes the desired open reading frame. Upon receipt, our molecular biology team performs an immediate integrity check—sequence-to-sequence alignment for electronic files, restriction fingerprint and Sanger snapshot for physical DNAs, or reverse-transcription-PCR for mRNA templates—thereby ensuring that every downstream step proceeds from an authenticated substrate. You are relieved of cloning logistics; simply transmit the material and we convert it into an endotoxin-free, transfection-grade IDLV transfer plasmid tailored to your specification.

• Golden-Gate Multiplex Assembly

To assemble the final construct we deploy either Golden-Gate or Gibson methodologies according to cassette complexity and repeat content. Golden-Gate is generally preferred over other methods for modular, multi-fragment designs, as it allows for concurrent, scar-less integration of multiple, independent components, such as promoters, ORFs and regulatory elements, all in the same reaction, without iterative cloning steps. When working with sequences that have high repeat content of RNA motifs or high GC content, Gibson assembly is also a good choice for seamless, isothermal joining, with less possibility of recombination-mediated deletion. Each of these methods is performed under uniform, internally-validated reaction conditions that ensure directionality and reading-frame integrity.

• Long-Read ITR/LTR Scan and Endotoxin-Clear Maxi-Prep

Long-read sequencing is done over the full ITR and LTR regions (regions that have long been thought to be recalcitrant to short-read methods) to ensure that no deletions, concatemerizations or point mutations occurred in the propagation of these elements in recombination-deficient E. coli. This step is necessary as single-base changes in the terminal repeats can affect vector genome packaging or reverse transcription. A complete, circular consensus map is generated and delivered to you as part of the documentation packet, providing absolute certainty of construct identity. Selection stringency is doubled by employing dual antibiotic cassettes (e.g., ampicillin plus kanamycin) cloned in tandem but flanked by distinct transcriptional terminators. This design minimizes the emergence of satellite colonies and virtually abolishes plasmid backbone rearrangements during high-density culture. Post-construction, plasmid DNA is purified via anion-exchange chromatography followed by endotoxin removal under endotoxin-controlled buffers, routinely yielding endotoxin loads compatible with sensitive primary cell transfection and in vivo administration without further processing.

• Electronic Data Package

As your project moves through the pipeline, you can access updates through a secure customer portal: gel pictures of intermediate assemblies, next-generation sequencing quality scores, and a final vector map in GenBank format. Your finished plasmid is shipped in aliquots that are pre-quantified by spectrophotometry and fluorescence-based double-strand DNA assays, with a complete certificate that includes cloning strategy, antibiotic resistance profile, and sequencing coverage. By entrusting the molecular build to our platform, you conserve valuable laboratory resources and accelerate the transition from design concept to functional IDLV production.

High-titer Production

To spare our clients the burden of in-house upstream development, Creative Biolabs offer a fully integrated production platform that begins the moment your sequence-verified plasmid enters our facility. The entire process is conducted in serum-free suspension cultures of HEK293T cells adapted to chemically defined medium, eliminating the variability associated with serum lots and simplifying downstream purification. Cultures are expanded in single-use bioreactors that accommodate flexible working volumes; this single-use strategy removes cross-contamination risk and allows rapid change-over between projects, ensuring that your vector is never queued behind another campaign.

• Triple-plasmid + chemical enhancer

Gene transfer is accomplished by a triple-transfection protocol in which the transfer plasmid, packaging construct, and envelope helper are delivered simultaneously. A minimally toxic transfection enhancer is co-administered for stabilization of DNA–lipid complexes and to facilitate nuclear uptake. This results in a strong balance between cytoplasmic trafficking and cell viability. Since the enhancer is completely synthetic, no adventitious agents are introduced and it is compatible with both research grade and regulatory compliant workflows.

• Real-time metabolite suite

On-line metabolite sensors continuously record glucose, lactate, glutamine, and dissolved oxygen, providing a real-time window into culture health. These parameters feed directly into a design-of-experiments (DoE) algorithm that adjusts feed volume, aeration rate, and transfection stoichiometry during the run. Such dynamic optimization maximizes the fraction of packaging-competent vector genomes while minimizing the accumulation of non-infectious particles, giving you a higher ratio of functional to total capsids without additional manual intervention.

• DoE-driven optimization

By merging serum-free suspension culture with real-time DoE control, we routinely achieve lentiviral supernatants that meet or exceed the functional titers demanded by modern pre-clinical applications, all without exposing your project to the delays and capital costs of stainless-steel equipment. The clarified harvest is immediately forwarded to downstream purification, providing a seamless transition from cell culture to final vector product and allowing you to focus on biology rather than bioprocess.

Advanced Purification

To free investigators from the burden of developing costly high-grade downstream processes, Creative Biolabs provides an end-to-end purification pipeline that begins with the clarified viral harvest and ends with an endotoxin-controlled, ready-to-use vector stock. The scheme is built around three orthogonal steps—anion-exchange capture, nuclease polishing, and size-exclusion chromatography—each selected for its ability to remove specific impurity classes while preserving infectious particles.

• Anion-exchange capture

Anion-exchange capture exploits the high isoelectric point of the VSV-G envelope and the negative charge of host-cell DNA. Your harvested supernatant is loaded onto a rigid, high-flow resin that binds vector particles under moderate conductivity; contaminating genomic DNA, residual plasmid, and the majority of host proteins flow through. A quickly high-salt wash strips DNA that might otherwise co-elute with virions, yet remains gentle enough to maintain envelope integrity. This single step routinely achieves orders-of-magnitude reduction in both protein and nucleic acid load, providing a clean feed for subsequent enzymatic digestion.

• Universal nuclease digestion

Universal nuclease treatment is then performed under defined ionic strength and magnesium activity that maximize DNase/RNase activity without compromising viral membranes. The enzyme is later quenched and removed during downstream diafiltration, ensuring that no exogenous protein is introduced into the final drug product. This digestion step eliminates residual linear DNA fragments that could confound functional titer assays or trigger innate immune sensors in vivo.

• Size-exclusion polishing

Size-exclusion polishing separates intact vector particles from empty capsids, nuclease-digested oligonucleotides, and trace aggregates. A composite agarose-dextran matrix fractionates species strictly by hydrodynamic radius; full vector genomes elute ahead of empty particles, allowing collection of a narrow pool enriched for genome-containing virions. The column is calibrated daily with protein standards that bracket the expected elution volume of IDLV, guaranteeing reproducible cut-points from batch to batch.

• Full/empty verification and Final formulation

Throughout purification, full-versus-empty ratios are monitored by dual techniques: ultraviolet absorbance spectroscopy provides a rapid, non-invasive estimate of nucleic acid content relative to total protein, while negative-stain transmission electron microscopy offers visual confirmation of particle morphology and internal genome density. These orthogonal measurements are documented in the certificate of analysis, giving you quantitative assurance of particle quality without additional in-house testing.

Comprehensive QC Analytics – Every Quality Attribute You Need

To eliminate the burden of in-house assay development and regulatory documentation, we provide an integrated analytics package that interrogates each lot at the genomic, protein, functional, and safety levels. Upon completion, you receive a single, indexed certificate summarizing orthogonal data and concise scientific interpretations—ready for institutional review, or grant submission.

• Functional genome titer

Functional genome titer is quantified by digital PCR. Your vector RNA genome is reverse-transcribed and probed against a single-copy standard, ensuring that only encapsidated, reverse-transcription-competent RNA is scored. The resulting value is reported as transduction units per volume, providing a direct input for dose–response calculations without further normalization. Physical particle count is obtained by p24 capsid ELISA. By comparing the functional titer to total p24, we calculate a particle-to-infectivity ratio—an internal quality benchmark that flags over-sheared or empty-rich preparations before they reach your laboratory.

• Replication-competent lentivirus (RCL)

RCL is assayed by vector-specific qPCR targeting the packaging signal and gag region. The assay is conducted on producer supernatant and on the final formulated bulk, guaranteeing that recombination events leading to mobilization are undetectable within the sensitivity limits of the method. Purity and impurity profiling begins with SDS-PAGE under reducing conditions, silver-stained to visualize the three major capsid bands (p24, p17, p7). Densitometry confirms the correct stoichiometry and reveals host-cell protein contamination. Residual host-cell DNA and protein are further quantified by threshold immunoenzymatic and qPCR methods; clearance factors are reported relative to the crude harvest, demonstrating robust impurity removal.

• Sterility and endotoxin

Sterility and endotoxin are assessed via compendial assays adapted to viral vectors. Endotoxin is measured by kinetic chromogenic Limulus amebocyte lysate, while sterility is confirmed by direct inoculation of both anaerobic and aerobic media. Mycoplasma presence is interrogated with a nucleic-acid amplification test, ensuring that common cell-culture contaminants are absent. All raw data traces—electropherograms, standard curves, gel images—are provided as electronic appendices. Where applicable, we append a brief scientific commentary explaining how each metric aligns with accepted thresholds for pre-clinical or clinical material, converting analytical numbers into actionable context for your downstream experiments.

Comparative Advantage – Why Choose Us?

Scientific Rigor

Our platform is steered by scientists who helped author the very literature on which your protocols rely. With a core team averaging more than a decade of lentivirus-focused research, we contribute peer-reviewed methodologies that have become industry benchmarks. When you outsource to Creative Biolabs, you gain access to this intellectual capital in real time: every vector design is cross-checked against empirically determined rules for promoter strength, payload stability, and integration-block fidelity, ensuring that the material arriving at your bench behaves exactly as described in published reports.

Speed

We recognize that grant deadlines and competitive filings wait for no one. Our standard service is configured for an aggressively compressed timeline that still accommodates full phase-appropriate QC, while a dedicated turbo track exists for early-discovery work where speed outweighs extensive documentation. Both routes share the same automated workflows and electronic project management portals, so you can monitor, download, and approve data packages 24 hours a day without administrative delay.

Flexibility

Whether you need a pilot-scale aliquot sufficient for a single in vitro screen or a multi-liter lot destined for a tox study, the manufacturing pathway is identical up to the final formulation step. This continuity eliminates re-validation surprises and allows seamless scale-up without re-cloning or re-optimizing titration assays. Volumes, buffer compositions, and even fill configurations can be customized on a per-project basis, accommodating unique administration devices or pre-clinical model constraints.

Quality

Our facility operates segregated suites that map to expectations, each supported by quality management systems that have been externally audited and certified. Critical raw materials are sourced with full traceability, and equipment qualification folders are available for regulatory review. By aligning the level of quality oversight with the intended use of the vector, we prevent both under-documenting (a regulatory risk) and over-documenting (an unnecessary cost), delivering exactly the degree of assurance your current stage demands.

Client Case Studies & Testimonials

"We had to reinvent episomal delivery from first principles when an intractable iPSC-derived cortical culture proved refractory to every integrase-competent vector we tried. We gave up nothing more than a SYN1 promoter FASTA and in return were given an IN(D64V) integration-deficient lentivector (from Creative Biolabs), whose cPPT/CTS flap had been elongated for nuclear import and whose LTRs were donning neuron-specific miR masks. The resulting stock transduced >90 % of MAP2-positive cells, was as episomal as a plasmid, and expressed the 6-kb calcium sensor for weeks without silencing. The precision of the design continues to elicit appreciative nods—and the occasional soft whistle—during our group meeting slide decks."

—Dr. Kenji Tanaka, Senior Investigator, Synaptic Engineering Unit

"The translational application we wanted to address with our brief required a vector to carry a 7-kb hypoxia reporter through a hypoxic tumor bulk and not integrate in the adjacent stroma. Creative Biolabs started from an IN(D64V) backbone, added a Shield-1 responsive destabilization domain, and cloaked the transgene in insulated, self-inactivating LTRs. The resulting particles traversed the hypoxic core with blockbuster elegance, demonstrated episomal stability across multiple patient-derived organoid lines, and had undetectable off-target integration. By eliminating months of integration-site curation, we compressed the development timeline and delivered the manuscript ahead of schedule—an efficiency gain that underscores the platform's technical maturity."

—Dr. Priya Natarajan, Director, Translational Neuro-Oncology

"Caught in an arms race with a recalcitrant hematopoietic stem cell line (refractory to all integrase-proficient vectors), we were at a point where we had to start thinking about gene delivery from first principles. We offered nothing more exotically sophisticated than a CD34 epitope FASTA, and we later received a Creative Biolabs-supplemented integration-defective lentivector whose nuclear import peptide had been optimized by a single-residue mutation and whose episomal backbone was adorned with lineage-specific miR veils. The resulting producer grew like a ghost through the marrow microenvironment, transduced the stem cell compartment without affecting differentiated progeny, and had an episome landscape as stable as centromeric chromatin. We initiated engraftment analyses in short time of vector arrival—an expeditionary pace that redefines turnaround efficiency in hematopoietic reconstitution workflows."

—Prof. Ana O'Neill, Principal Scientist, Stem Cell Gene Therapy Division

FAQ

References

1. Nemirov, Kirill, et al. "Lentiviral vectors as a vaccine platform against infectious diseases." Pharmaceutics 15.3 (2023): 846. https://doi.org/10.3390/pharmaceutics15030846.
2. Distributed under Open Access license CC BY 4.0, without modification.