summary
Simple co-injection of Cre recombinase with HIV-TAT peptide successfully delivered Cre to mouse nerve cells, and Cre's delivery successfully activated the expression of reporter genes in neurons and astrocytes in the cerebral cortex without causing tissue damage, and its transduction efficiency was comparable or better
than that of commonly used adeno-associated viruses.
Research data suggest that transport peptides mediate endoplasmic leakage and cytoplasmic escape of potent endocytosis Cre cells
.
Therefore, the peptide acts in trans and does not need to bind to the payload, greatly simplifying sample preparation
.
In addition, transport peptides are composed entirely of natural amino acids and are therefore easily degraded and processed
by cells.
This method will facilitate applications that require transient introduction of proteins into cells in vivo
.
The article was published in the new issue of SCIENCE ADVANCES
.
Brief introduction
Understanding the physiology of the central nervous system, especially the brain, is one of the most pressing challenges in the medical community today
.
Studying how cells interact in time and space, in health or disease, and in the context of neural networks is complex
.
It requires new research tools that allow the delivery of probes
that can report cells within the cell.
Intracranial stereotactic injection of viruses, especially adeno-associated viruses (AAVs), is often used to study the manipulation and understanding
of central nervous system (CNS) cells in vivo.
Genes transferred by these viruses can be fluorescent reporters, or genome editing tools used to monitor and regulate cellular processes
.
AAV still has some limitations, including relatively low packaging capacity, slow initiation of transgene expression, low transduction efficiency in some cases, possible integration into the host genome, and induced neuroinflammatory responses
.
In addition, the continuous expression of transferred genes is beneficial for some applications, but problematic
for others.
Gene editing experiments are like this, where the persistence of gene-editing mechanisms in cells leads to off-target genomic modifications
.
The expression of transcription factors also often requires both transient and transient control
.
Direct delivery of proteins can avoid these problems
.
Proteins can initiate intracellular activity
without transcription/translation delays.
The protein's half-life within the cell is also limited, so long-term effects
can be avoided.
A major limitation of the use of proteins as intracellular delivery payloads is the lack of current technologies
for the efficient and low-toxicity introduction of these macromolecules into the cytoplasm.
Many deliverers are able to facilitate the endocytosis of proteins into cells, yet the payload of proteins is usually still trapped in endosomes
.
Therefore, the payloads of these proteins cannot reach cytoplasmic or nuclear targets and cannot induce the required biological activity
.
Peptides
that can selectively penetrate late endocytosis (LE, also known as secondary endocytosis) membranes have been developed.
These peptides include a dimer dfTAT of the cell-penetrating peptide (CPP) TAT
.
When topically administered to cells, both the dfTAT and protein payload accumulate into the endocytotic chamber and then transported together to the late endocytosis, where dfTAT mediates membrane permeability and allows the protein active ingredient to escape into the
cytoplasm.
It is important to note that the use of dfTAT does not require coupling or binding to payloads
.
As long as both molecules are swallowed up intracellular to reach the same endocytosis, cytoplasmic delivery is
successful.
This approach is significantly different
from the strategy of vector systems to encapsulate payloads and carry them to cells such as liposomes and nanoparticles.
The main benefits of this approach include simple sample preparation, controllable payloads, and the ability to deliver chemically and functionally unchanged "original" proteins
.
Although this strategy is useful in vitro cell culture environments, it is unclear whether it can function in vivo because the delivery agent and "cargo" may diffuse to each other in the tissue and reach different cells
separately.
In addition, a major difference between in vitro tissue culture and in vivo application is the time
when cells are exposed to delivery agents and "cargo".
In tissue culture, the concentration of the delivery agent and the cargo is relatively constant, while in vivo, these concentrations may change
due to the inherent pharmacokinetic properties.
It can be expected that this difference will have a significant impact on
delivery and toxicity.
Given these key differences between in vitro and in vivo methods, the researchers aim in this study to explore the effectiveness of this cell-penetrating peptide in the case of stereotactic intracranial injection, establishing the feasibility
of a protein delivery method that is as effective as AAV transduction.
This technique allows the use of protein-based tools and probes to manipulate and study in vivo histophysiology
.
The potential of this approach as a therapeutic application will be explored in the future, as it has unique advantages
over viral delivery and current state-of-the-art payload delivery.
outcomeOne of the benefits of protein transduction is that they act quickly within the cell and are subsequently cleared without long-term harmful effects
.
In this case a delivery tool should be used, which degrades rapidly once it enters the cell with little residue
.
dfTAT is a peptide
that degrades rapidly during and after cell infiltration.
The initial dfTAT contained two copies of carboxytetramethylrhodamine (TMR, a fluorophore) that allowed for microscopy observation of cell penetration processes and mechanistic studies
.
To avoid the transport process and the accumulation of fluorescent material after degradation, the researchers tried to develop dfTAT analogues – which do not contain cell-foreign substances and break down into natural amino acids.
However, preliminary tests have shown that simply removing TMR from dfTAT yields no transitivity, and they speculate that the relative hydrophobicity of TMR may be related
to the disruptive activity of the endoplasmic membrane of dfTAT.
They synthesized an analogue library with a general structure of d(X)TAT, where "X" is a hydrophobic residue, including short hydrophobic peptide sequences, peptide acceleration sequence Pas (amino acid sequence: FFLIP) and a peptide derived from human papillomavirus type 33 capsid protein L2 (amino acid sequence: YFIL) – previously identified to enhance the cellular permeability
of CPPs.
To assess the transport activity of these peptides (which are not directly visible under fluorescence microscopy and are not directly observed as dfTAT), TMR-k5 is used as an indicator in conjunction with d(X)TAT (to prevent proteolytic degradation of peptides in cells, with d-lysine residues, indicated in lowercase letters).
TMR-k5 is easily endocytosized, but lacks its own endocytosis escape activity, once TMR-k5 enters the cell cytoplasm will be distributed throughout the cytoplasm, reaching the nucleus, staining the nucleoli
.
Using this unique feature that confirms the intracellular pathway, the number of cells showing fluorescent nuclei in the presence of d(X)TAT reagents is counted
.
The percentage of cells stained with TMR-k5-stained nuclei is used as a measure of the relative transport efficiency of each d(X)TAT peptide (the detection of TMR-k5 itself depends on the detection threshold of the microscope used and the concentration of TMR-k5 used during incubation).
The SYTOX rejection test is used to assess peptide toxicity and exclude dead cells
.
The test results showed that d(LL) TATs containing two continuous leucine residues reproduced the transport activity of dfTAT, while some peptides (X=WW, FFLIP, and YFIL) were generally 5-10 times
higher than dfTAT activity.
The d(X)TAT peptide loses activity
in its monomer form.
This suggests that, from a structural-activity point of view, both the "X" position and dimer action regulate the transport activity
of the peptide.
Notably, the d(X)TAT peptide shows little cytotoxicity
at concentrations that achieve cytoplasmic transport in a high percentage of cells.
The authors selected d(LL)TAT and d(WW)TAT as representative members of d(X)TAT peptides with medium and high transport activity for follow-up studies
.
To further validate these results, the authors evaluated the delivery effect
of these transmissive peptides on the gene-modifying enzyme Cre recombinase.
The results showed that d(X)TAT did not change the endocytosis uptake level of TAT-Cre, but only caused the protein to be redistributed from the endocytosis to the nucleus
.
The d(X)TAT peptide enables TAT-Cre to transport TAT-Cre into the cytoplasm and nucleus of cells in vivo at similar levels to viral transduction Cre
.
discussThe authors propose a new non-viral method that can efficiently deliver proteins to cells in the
central nervous system.
This method consists of a polypeptide
that is simply mixed with the protein payload.
The authors used the payload Cre recombinase as a tool to spatially initiate gene expression in genetically modified organisms, demonstrating that this method is as effective
as the AAV gene delivery protocol in activating tdTom expression.
This approach offers some potential benefits: (i) it allows for a rapid start of intracellular biological activity (e.
g.
, Cre enzymes are delivered in their active form and quickly induce recombination after entering the cell, while AAV2-Cre requires a period of delay for the Cre recombinant enzyme to be expressed from the cell by the Cre gene).
(ii) Protein is delivered directly and transiently into cells, potentially avoiding unnecessary non-targeted and knock-on effects
caused by prolonged expression in the context of transcription factors or gene-editing tools.
(iii) Through standard solid-phase peptide synthesis and recombination techniques, delivery peptides and protein payloads can be produced directly, which greatly reduces the production time and costs
associated with viral strategies.
(iv) The delivery method does not involve potential risks
associated with host genomic DNA integration.
Note that this approach offers an alternative, not a replacement
, to AAV.
Transporting non-AAV packaged payloads (e.
g.
, DNA >5 kb, mRNA or long non-coding RNAs, large transcriptional regulators, etc.
), various proteins, peptides, oligonucleotides, small molecules that do not penetrate cells, and large nanoparticles (about 100 nm in diameter) have been successfully transported into cells by dfTAT co-precipitation
.
Notably, previous studies have also enabled gene editing of CNS by delivering Cas9 ribonucleoprotein or Cre recombinase in vivo, however these methods require modifying proteins with fusion tags that may interfere with protein function and using lipid-based transfectors, which may persist intracellular and trigger multiple cellular responses
.
In contrast, the CPPs used here can degrade rapidly after delivery (dfTAT has been shown to have little effect on cell physiology), and the protein cargo does not need to be modified, and these characteristics of the transmissive peptide are expected to bring benefits
for future research in vivo gene editing applications.
(See the original article for more discussion).
